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.

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.

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.

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.

Biodegradation of metal-based ultra-small nanoparticles: A combined approach using TDA-ICP-MS and CE-ICP-MS.

The knowledge of the fate of metal-containing nanoparticles in biological media in aqueous media is of utmost importance for the future use of these promising theranostic agents for clinical applications. A methodology based on the combination of TDA-ICP-MS and CE-ICP-MS was applied to study the degradation pathway of AGuIX, a phase 2 clinical ultrasmall gadolinium-containing nanoparticle. Nanoparticle size measurements and gadolinium speciation performed in different media (phosphate buffer, urine and serum) demonstrated an accelerated dissolution of AGuIX in serum, without any release of free gadolinium for each medium.

Taylor Dispersion Analysis Coupled to Inductively Coupled Plasma-Mass Spectrometry for Ultrasmall Nanoparticle Size Measurement: From Drug Product to Biological Media Studies.

During past decade, special focus has been laid on ultrasmall nanoparticles for nanomedicine and eventual clinical translation. To achieve such translation, a lot of challenges have to be solved. Among them, size determination is a particularly tricky one. In this aim, we have developed a simple hyphenation between Taylor dispersion analysis and inductively coupled plasma-mass spectrometry (ICP-MS). This method was proven to allow the determination of the hydrodynamic radius of metal-containing nanoparticles, even for sizes under 5 nm, with a relative standard deviation below 10% (with a 95% confidence interval) and at low concentrations. Moreover, its specificity provides the opportunity to perform measurements in complex biological media. This was applied to the characterization of an ultrasmall gadolinium-containing nanoparticle used as a theranostic agent in cancer diseases. Hydrodynamic radii measured in urine, cerebrospinal fluid, and undiluted serum demonstrated the absence of interaction between the particle and biological compounds such as proteins.

Radiosensitization Effect of AGuIX, a Gadolinium-Based Nanoparticle, in Nonsmall Cell Lung Cancer.

Radiotherapy is the main treatment for cancer patients. A major concern in radiotherapy is the radiation resistance of some tumors, such as human nonsmall cell lung cancer. However, the radiation dose delivered to the tumors is often limited by the possibility of collateral damage to surrounding healthy tissues. A new and efficient gadolinium-based nanoparticle, AGuIX, has recently been developed for magnetic resonance imaging-guided radiotherapy and has been proven to act as an efficient radiosensitizer. The amplified radiation effects of AGuIX nanoparticles appear to be due to the emission of low-energy photoelectrons and Auger electron interactions. We demonstrated that AGuIX nanoparticles exacerbated radiation-induced DNA double-strand break damage and reduced DNA repair in the H1299 nonsmall cell lung cancer cell line. Furthermore, we observed a significant improvement in tumor cell damage and growth suppression, under radiation therapy, with the AGuIX nanoparticles in a H1299 mouse xenograft model. This study paves the way for research into the radiosensitization mechanism of AGuIX nanoparticles and provides a scientific basis for the use of AGuIX nanoparticles as radiosensitizing drugs.

AGuIX® from bench to bedside-Transfer of an ultrasmall theranostic gadolinium-based nanoparticle to clinical medicine.

AGuIX® are sub-5 nm nanoparticles made of a polysiloxane matrix and gadolinium chelates. This nanoparticle has been recently accepted in clinical trials in association with radiotherapy. This review will summarize the principal preclinical results that have led to first in man administration. No evidence of toxicity has been observed during regulatory toxicity tests on two animal species (rodents and monkeys). Biodistributions on different animal models have shown passive uptake in tumours due to enhanced permeability and retention effect combined with renal elimination of the nanoparticles after intravenous administration. High radiosensitizing effect has been observed with different types of irradiations in vitro and in vivo on a large number of cancer types (brain, lung, melanoma, head and neck…). The review concludes with the second generation of AGuIX nanoparticles and the first preliminary results on human.

Direct determination of molecular weight distribution of calf-thymus DNAs and study of their fragmentation under ultrasonic and low-energy infrared irradiations. A charge detection mass spectrometry investigation.

RATIONALE: Calf-thymus (CT-DNA) is widely used as a binding agent. The commercial samples are known to be "highly polymerized DNA" samples. CT-DNA is known to be fragile in particular upon ultrasonic wave irradiation. Degradation products could have dramatic consequences on its bio-sensing activity, and an accurate determination of the molecular weight distribution and stability of commercial samples is highly demanded. METHODS: We investigated the sensitivity of charge detection mass spectrometry (CDMS), a single-molecule MS method, both with single-pass and ion trap CDMS ("Benner" trap) modes to the determination of the composition and stability (under multiphoton IR irradiation) of calf-thymus DNAs. We also investigated the changes in molecular weight distributions in the course of sonication by irradiating ultrasonic waves to CT-DNA. RESULTS: We report, for the first time, the direct molecular weight (MW) distribution of DNA sodium salt from calf-thymus revealing two populations at high (~10 MDa) and low (~3 MDa) molecular weights. We evidence a transition between the high-MW to the low-MW distribution, confirming that the low-MW distribution results from degradation of CT-DNA. Finally, we report also IRMPD experiments carried out on trapped single-stranded linear DNAs from calf-thymus allowing extraction of their activation energy for unimolecular dissociation. CONCLUSIONS: We show that single-pass CDMS is a direct, efficient and accurate MS-based approach to determine the composition of calf-thymus DNAs. Furthermore, ion trap CDMS allows us to evaluate the stability (both under multiphoton IR irradiation and in the course of sonication by irradiating ultrasonic wave) of calf-thymus DNAs.

One-pot direct synthesis for multifunctional ultrasmall hybrid silica nanoparticles.

Ultrasmall silica nanoparticles (NPs), having hydrodynamic diameters under 10 nm are promising inorganic platforms for imaging and therapeutic applications in medicine. Herein is described a new way for synthesizing such kind of NPs in a one-pot scalable protocol. These NPs bear DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) ligands on their surface that can chelate different metals suitable for a wide variety of biomedical applications. By varying the ratio of the precursors, the hydrodynamic diameters of the particles can be controlled over the range of 3 to 15 nm. The resulting NPs have been characterized extensively by complementary techniques like dynamic light scattering (DLS), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), mass spectrometry (MS), phosphorescence titration, photophysical measurements, relaxometry and elemental analysis to elucidate their structures. Chelation of gadolinium (Gd) allowed its use as an effective intravenous contrast agent in MRI and was illustrated in mice bearing colorectal CT26 tumors. The new particle appears to sufficiently accumulate in the tumors and efficiently clear out of animal bodies through kidneys. This new synthesis is an original, time/material-saving and very flexible process that can be applied for creating versatile ultrasmall multifunctional nanomedicines.

Amyloid-like aggregates formation by blood plasma fibronectin.

Fibronectin (FN) is a multifunctional glycoprotein of the extracellular matrix (ECM) playing critical roles in physiological and pathological cell processes like adhesion, migration, growth, and differentiation. These various functions of FN are modulated by its supramolecular state. Indeed, FN can polymerize into different types of assemblies like fibrils and aggregates. However, the mechanism of polymerization and the effects of such assemblies on cell behaviors still remain to be elucidated. Here we show that upon irreversible thermal denaturation, human blood plasma fibronectin forms high molecular weight aggregates. These compact and globular aggregates show amyloid features: they are stabilized by intermolecular b-sheets, they bind Thioflavin T and they are resistant to reducing and denaturing agents. Their characterization by electrospray ionization charge detection mass spectrometry shows that two populations can be distinguished according to the mass and charge density. Despite their amyloid features and the presence of hydrophobic patches on their surface, these aggregates are not toxic for cells. However, their binding abilities to gelatin and RGD are drastically decreased compare to native FN, suggesting possible effects on ECM-cell interactions.

Tuning the architectural integrity of high-performance magneto-fluorescent core-shell nanoassemblies in cancer cells.

High-density nanoarchitectures, endowed with simultaneous fluorescence and contrast properties for MRI and TEM imaging, have been obtained using a simple self-assembling strategy based on supramolecular interactions between non-doped fluorescent organic nanoparticles (FON) and superparamagnetic nanoparticles. In this way, a high-payload core-shell structure FON@mag has been obtained, protecting the hydrophobic fluorophores from the surroundings as well as from emission quenching by the shell of magnetic nanoparticles. Compared to isolated nanoparticles, maghemite nanoparticles self-assembled as an external shell create large inhomogeneous magnetic field, which causes enhanced transverse relaxivity and exacerbated MRI contrast. The magnetic load of the resulting nanoassemblies is evaluated using magnetic sedimentation and more originally electrospray mass spectrometry. The role of the stabilizing agents (citrate versus polyacrylate anions) revealed to be crucial regarding the cohesion of the resulting high-performance magneto-fluorescent nanoassemblies, which questions their use after cell internalization as nanocarriers or imaging agents for reliable correlative light and electron microcopy.

Optical extinction and scattering cross sections of plasmonic nanoparticle dimers in aqueous suspension.

Absolute extinction and scattering cross sections for gold nanoparticle dimers were determined experimentally using a chemometric approach involving singular-value decomposition of the extinction and scattering spectra of slowly aggregating gold nanospheres in aqueous suspension. Quantitative spectroscopic data on plasmonic nanoparticle assemblies in liquid suspension are rare, in particular for particles larger than 40 nm, and in this work we demonstrate how such data can be obtained directly from the aggregating suspension. Our method can analyse, non invasively, the evolution of several sub-populations of nanoparticle assemblies. It may be applied to other self-assembling nanoparticle systems with an evolving optical response. The colloidal systems studied here are based on 20, 50 and 80 nm gold nanospheres in aqueous solutions containing sodium lipoate. In these systems, the reversible dimerisation process can be controlled using pH and ionic strength, and this control is rationalised in terms of DLVO theory. The dimers were identified in suspension by their translational and rotational diffusion through scattering correlation spectroscopy. Moreover, their gigadalton molecular weight was measured using electrospray charge-detection mass spectrometry, demonstrating that mass spectrometry can be used to study nanoparticles assemblies of very high molecular mass. The extinction and scattering cross sections calculated in the discrete-dipole approximation (DDA) agree very well with those obtained experimentally using our approach.

Mass Determination of Entire Amyloid Fibrils by Using Mass Spectrometry.

Amyloid fibrils are self-assembled protein structures with important roles in biology (either pathogenic or physiological), and are attracting increasing interest in nanotechnology. However, because of their high aspect ratio and the presence of some polymorphism, that is, the possibility to adopt various structures, their characterization is challenging and basic information such as their mass is unknown. Here we show that charge-detection mass spectrometry, recently developed for large self-assembled systems such as viruses, provides such information in a straightforward manner.

Coupling of size-exclusion chromatography with electrospray ionization charge-detection mass spectrometry for the characterization of synthetic polymers of ultra-high molar mass.

RATIONALE: Coupling size-exclusion chromatography (SEC) with mass spectrometry (MS) allowed generation of a SEC calibration curve based on the analyte itself, which is more reliable than calibration based on non-related standards and faster than the use of the multiple detection mode. However, such SEC/MS couplings were limited to rather small synthetic polymers. METHODS: Based on the concept of image current detection, charge-detection mass spectrometry (CDMS) coupled to electrospray ionization (ESI) is a useful method for weighing macro-ions from compounds with masses higher than one megadalton (MDa). Using columns designed to allow analysis of synthetic polymers of over 15 million Dalton in mass, performance of the SEC/ESI-CDMS coupling was evaluated for polyacrylamide (PAM, 5-6 MDa) and a poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS, 2 MDa). RESULTS: The SEC/ESI-CDMS profiles were first compared with SEC-UV profiles: the systematic shift in retention time was assigned to the slightly different geometries of the two instrumental systems. The SEC/ESI-CDMS data were then compared with results obtained after the direct infusion of each sample into the ESI source. Both the shape of the molecular weight distribution and the mass values were similar with or without separation prior to ESI, and these values were consistent with data provided by the sample supplier. CONCLUSIONS: SEC/MS incorporating an online ESI-CDMS coupling was shown to be a rapid and efficient technique for the analysis of water-soluble synthetic polymers with ultra-high molecular mass in the megadalton range. The coupling also afforded an attractive solution for SEC calibration without the use of external standards.

Testing the vesicular morphology to destruction: birth and death of diblock copolymer vesicles prepared via polymerization-induced self-assembly.

Small angle X-ray scattering (SAXS), electrospray ionization charge detection mass spectrometry (CD-MS), dynamic light scattering (DLS), and transmission electron microscopy (TEM) are used to characterize poly(glycerol monomethacrylate)55-poly(2-hydroxypropyl methacrylate)x (G55-Hx) vesicles prepared by polymerization-induced self-assembly (PISA) using a reversible addition-fragmentation chain transfer (RAFT) aqueous dispersion polymerization formulation. A G55 chain transfer agent is utilized to prepare a series of G55-Hx diblock copolymers, where the mean degree of polymerization (DP) of the membrane-forming block (x) is varied from 200 to 2000. TEM confirms that vesicles with progressively thicker membranes are produced for x = 200-1000, while SAXS indicates a gradual reduction in mean aggregation number for higher x values, which is consistent with CD-MS studies. Both DLS and SAXS studies indicate minimal change in the overall vesicle diameter between x = 400 and 800. Fitting SAXS patterns to a vesicle model enables calculation of the membrane thickness, degree of hydration of the membrane, and the mean vesicle aggregation number. The membrane thickness increases at higher x values, hence the vesicle lumen must become smaller if the external vesicle dimensions remain constant. Geometric considerations indicate that this growth mechanism lowers the total vesicle interfacial area and hence reduces the free energy of the system. However, it also inevitably leads to gradual ingress of the encapsulated water molecules into the vesicle membrane, as confirmed by SAXS analysis. Ultimately, the highly plasticized membranes become insufficiently hydrophobic to stabilize the vesicle morphology when x exceeds 1000, thus this PISA growth mechanism ultimately leads to vesicle "death".

Multiphoton dissociation of electrosprayed megadalton-sized DNA ions in a charge-detection mass spectrometer.

Charge detection mass spectrometry in combination with a linear electrostatic ion trap coupled to a continuous wavelength infrared CO2 laser has been used to study the multiphoton dissociation of DNA macromolecular ions. Samples, with masses ranging from 2.23 to 31.5 MDa, include single strand circular M13mp18, double strand circular M13mp18, and double strand linear LambdaPhage DNA fragments. Their activation energies for unimolecular dissociation were determined. Activation energy values slightly increase as a function of the molecular weight. The most important result is the difference between the fragmentations observed for hybridized double-strands and dimers of single strands.

Correlation between the charge of polymer particles in solution and in the gas phase investigated by zeta-potential measurements and electrospray ionization mass spectrometry.

The relationship between the effective charge of polymer nanoparticles (PNP) in solution and the charge states of ionized particles produced in the gas phase by electrospray ionization was investigated. Charge detection mass spectrometry was used to measure both the mass and charge of individual electrosprayed ions. The effective charges extracted from the measured zeta-potential of PNPs in solution are partially correlated with the average values of charge of PNPs in the gas phase. The correlation between the magnitude of charging of PNPs ions produced in the gas phase with the PNPs surface charge in solution demonstrates that the mass spectrometry-based analysis described in this work is an alternative and promising way for a fast and systematic characterization of charges on colloidal particles.

The charging of micellar nanoparticles in electrospray ionization.

Charging of nanoparticles through electrospray has scarcely been explored. Spherical nanometer-sized amphiphilic block copolymer nanoparticles with diameters ranging from ∼65 to ∼150 nm were electrosprayed and analysed by charge detection spectrometry. Herein, we explore the charging of these micellar nano-objects by conducting a thorough study in different solvents, including pure water, and upon the addition of "supercharging" agents. The charge (z) of micellar nanoparticles electrosprayed from water solution is compared to the Rayleigh's limiting charge (z(R)) of a charged water droplet of the same dimensions. An average ratio (z/z(R)) of 0.6-0.65 is observed for the micellar macro-ions, supporting the charge residue mechanism, where the number of charges available to the micellar macro-ion is limited by the number of charges on the nanodroplet, which is a function of the surface tension of the solvent. Also we show the possibility of increasing the charging of micellar nanoparticles in the negative mode by adding organic bases (in particular piperidine) to water/methanol solutions.

Direct Molar Mass Determination of Self-Assembled Amphiphilic Block Copolymer Nanoobjects Using Electrospray-Charge Detection Mass Spectrometry.

Charge detection mass spectrometry (CD-MS) combined with electrospray ionization was used to determine, in a direct way and for the first time, the molar mass of self-assembled amphiphilic block copolymer nanoobjects prepared via living radical emulsion polymerization. CD-MS supplies enough data for calculating statistically significant measurements of the mass of particles in the megadalton to gigadalton range and their resulting mass distribution.