In Vivo Evaluation of Innovative Gadolinium-Based Contrast Agents Designed for Bioimaging Applications.

The aim of this study was the investigation of biochemical and histological changes induced in different tissues, as a result of the subcutaneous administration of Gd nanohydrogels (GdDOTA⸦CS-TPP/HA) in a CD-1 mouse strain. The nanohydrogels were obtained by encapsulating contrast agents (GdDOTA) in a biocompatible polymer matrix composed of chitosan (CS) and hyaluronic acid (HA) through the ionic gelation process. The effects of Gd nanohydrogels on the redox status were evaluated by measuring specific activities of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD), as well as oxidative stress markers, such as reduced glutathione (GSH), malondialdehyde (MDA), advanced oxidation protein products (AOPP), and protein-reactive carbonyl groups (PRCG), in the liver, kidney, and heart tissues. The nitrosylated proteins expression were analyzed with Western Blot and the serum biochemical markers were measured with spectrophotometric methods. Also, a histological analysis of CD-1 mouse tissues was investigated. These results indicated that Gd nanohydrogels could potentially be an alternative to current MRI contrast agents thanks to their low toxicity in vivo.

In Vitro Studies Regarding the Safety of Chitosan and Hyaluronic Acid-Based Nanohydrogels Containing Contrast Agents for Magnetic Resonance Imaging.

The aim of this study was to investigate the biocompatibility of contrast agents, such as gadolinium 1, 4, 7, 10 tetraazacyclo-dodecane tetraacetic acid (GdDOTA) and gadolinium dioctyl terephthalate (GdDOTP), encapsulated in a polymeric matrix containing chitosan and hyaluronic acid using RAW264.7 murine macrophages and human blood samples. The cell viability and cytotoxicity were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, while cell cycle analysis was determined in RAW264.7 cells using flow cytometry. The mitochondrial membrane potential (MMP), hemolytic index, complement activation, and thrombogenic potential of gadolinium (Gd) containing nanohydrogels were measured by fluorometric and spectrophotometric methods. Taken together, our results demonstrate the good bio- and hemocompatibility of chitosan-based nanohydrogels with the RAW264.7 cell line and human blood cells, suggesting that these could be used as injectable formulations for the magnetic resonance imaging diagnostic of lymph nodes.

Synthesis and Characterization of Conjugated Hyaluronic Acids. Application to Stability Studies of Chitosan-Hyaluronic Acid Nanogels Based on Fluorescence Resonance Energy Transfer.

Hyaluronic acid (HA) was functionalized with a series of amino synthons (octylamine, polyethylene glycol amine, trifluoropropyl amine, rhodamine). Sodium hyaluronate (HAs) was first converted into its protonated form (HAp) and the reaction was conducted in DMSO by varying the initial ratio (-NH2 (synthon)/COOH (HAp)). HA derivatives were characterized by a combination of techniques (FTIR, 1H NMR, 1D diffusion-filtered 19F NMR, DOSY experiments), and degrees of substitution (DSHA) varying from 0.3% to 47% were determined, according to the grafted synthon. Nanohydrogels were then obtained by ionic gelation between functionalized hyaluronic acids and chitosan (CS) and tripolyphosphate (TPP) as a cross-linker. Nanohydrogels for which HA and CS were respectively labeled by rhodamine and fluorescein which are a fluorescent donor-acceptor pair were subjected to FRET experiments to evaluate the stability of these nano-assemblies.

Targeting Ovarian Carcinoma with TSP-1:CD47 Antagonist TAX2 Activates Anti-Tumor Immunity.

TAX2 peptide is a cyclic peptide that acts as an orthosteric antagonist for thrombospondin-1 (TSP-1) interaction with CD47. TAX2 was first described for its anti-angiogenic activities and showed anti-cancer efficacy in numerous preclinical models. Here, we aimed at providing an extensive molecular characterization of TAX2 mode of action, while evaluating its potential in ovarian cancer therapy. Multidisciplinary approaches were used to qualify a TAX2 drug candidate in terms of stability, solubility and potency. Then, efficacy studies, together with benchmark experiments, were performed in relevant mouse models of ovarian carcinoma. TAX2 peptide appears to be stable and soluble in clinically relevant solvents, while displaying a favorable safety profile. Moreover, clinical data mining allowed for the identification of TSP-1 as a relevant pharmacological target in ovarian cancer. In mice, TAX2 therapy inhibits ovarian tumor growth and metastatic dissemination, while activating anti-cancer adaptive immunity. Interestingly, TAX2 also synergizes when administered in combination with anti-PD-1 immune checkpoint inhibitiors. Altogether, our data expose TAX2 as an optimized candidate with advanced preclinical characterization. Using relevant syngeneic ovarian carcinoma models, we highlighted TAX2's ability to convert poorly immunogenic tumors into ones displaying effective anti-tumor T-cell immunity.

Investigation of squalene-doxorubicin distribution and interactions within single cancer cell using Raman microspectroscopy.

Intracellular distribution of doxorubicin (DOX) and its squalenoylated (SQ-DOX) nanoparticles (NPs) form in murine lung carcinoma M109 and human breast carcinoma MDA-MB-231 cells was investigated by Raman microspectroscopy. Pharmacological data showed that DOX induced higher cytotoxic effect than SQ-DOX NPs. Raman data were obtained using single-point measurements and imaging on the whole cell areas. These data showed that after DOX treatment at 1 µM, the spectral features of DOX were not detected in the M109 cell cytoplasm and nucleus. However, the intracellular distribution of SQ-DOX NPs was higher than DOX in the same conditions. In addition, SQ-DOX NPs were localized into both cell cytoplasm and nucleus. After 5 µM treatment, Raman bands of DOX at 1211 and 1241 cm-1 were detected in the nucleus. Moreover, the intensity ratio of these bands decreased, indicating DOX intercalation into DNA. However, after treatment with SQ-DOX NPs, the intensity of these Raman bands increased. Interestingly, with SQ-DOX NPs, the intensity of 1210/1241 cm-1 ratio was higher suggesting a lower fraction of intercalated DOX in DNA and higher amount of non-hydrolyzed SQ-DOX. Raman imaging data confirm this subcellular localization of these drugs in both M109 and MDA-MB-231 cells. These finding brings new insights to the cellular characterization of anticancer drugs at the molecular level, particularly in the field of nanomedicine.

Transmembrane Peptides as a New Strategy to Inhibit Neuraminidase-1 Activation.

Sialidases, or neuraminidases, are involved in several human disorders such as neurodegenerative, infectious and cardiovascular diseases, and cancers. Accumulative data have shown that inhibition of neuraminidases, such as NEU1 sialidase, may be a promising pharmacological target, and selective inhibitors of NEU1 are therefore needed to better understand the biological functions of this sialidase. In the present study, we designed interfering peptides (IntPep) that target a transmembrane dimerization interface previously identified in human NEU1 that controls its membrane dimerization and sialidase activity. Two complementary strategies were used to deliver the IntPep into cells, either flanked to a TAT sequence or non-tagged for solubilization in detergent micelles. Combined with molecular dynamics simulations and heteronuclear nuclear magnetic resonance (NMR) studies in membrane-mimicking environments, our results show that these IntPep are able to interact with the dimerization interface of human NEU1, to disrupt membrane NEU1 dimerization and to strongly decrease its sialidase activity at the plasma membrane. In conclusion, we report here new selective inhibitors of human NEU1 of strong interest to elucidate the biological functions of this sialidase.

Biocompatibility of Gd-Loaded Chitosan-Hyaluronic Acid Nanogels as Contrast Agents for Magnetic Resonance Cancer Imaging.

Although the research on nanogels incorporating Gd chelates for theranostic applications has grown exponentially in recent years, knowledge about their biocompatibility is limited. We compared the biocompatibility of Gd-loaded hyaluronic acid-chitosan-based nanogels (GdCA⊂CS-TPP/HA) with two chitosan concentrations (2.5 and 1.5 mg·mL-1 respectively) using SVEC4-10 murine lymph node endothelial cells. The sulforhodamine B method and released lactate dehydrogenase (LDH) activity were used as cell viability tests. Reactive oxygen species (ROS), reduced glutathione (GSH) and malondialdehyde (MDA) were measured by spectrophotometric and fluorimetric methods. Nrf-2 protein expression was evaluated by Western blot analysis and genotoxicity by alkaline comet assay. After 24 h, the cells viability was not affected by all types and doses of nanogels. The increase of ROS induced a low decrease of GSH concentration and a time-dependent raise of MDA one was produced by citric GdDOTA⊂CS-TPP/HA with a chitosan concentration of 1.5 mg·mL-1, at the highest dose applied. None of the tested nanogels induced changes in Nrf-2 protein expression. A slight but significant genotoxic effect was caused only by citric GdDOTA⊂CS-TPP/HA where CS concentration was 1.5 mg·mL-1. Our results showed a better biocompatibility with lymph node endothelial cells for Gd-loaded hyaluronic acid-chitosan based nanogels with a concentration in chitosan of 2.5 mg·mL-1.

Synthesis and Characterization of PEGylated and Fluorinated Chitosans: Application to the Synthesis of Targeted Nanoparticles for Drug Delivery.

To synthesize chitosan nanoparticles (CS NPs), ionic gelation is a very attractive method. It relies on the spontaneous supramolecular assembly of cationic CS with anionic compounds, which leads to nanohydrogels. To extend ionic gelation to functionalized CS, the assessment of CS degree of substitution (DSCS) is a key step. In this paper, we have developed a hyphenated strategy for functionalized CS characterization, based upon 1H, DOSY and, when relevant, 1D diffusion-filtered 19F NMR spectroscopies. For that, we have synthesized two series of water-soluble CS via amidation of CS amino groups with mPEG2000-COOH or fluorinated synthons (TFB-COOH). The aforementioned NMR techniques helped to discriminate between ungrafted and grafted synthons and finally to determine DSCS. According to DSCS values, the selection of CS-mPEG2000 or CS-TFB copolymers can be made to obtain, in the presence of hyaluronic acid (HA) and tripolyphosphate (TPP), CS-mPEG2000-TPP/HA or CS-TFB-TPP/HA nanohydrogels suitable for drug delivery.

Tuning the composition of biocompatible Gd nanohydrogels to achieve hypersensitive dual T1/T2 MRI contrast agents.

A series of hydrogel nanoparticles incorporating MRI contrast agents (GdDOTP and MS325) as potential cross-linkers were elaborated by an easy and robust ionotropic gelation process. By this process, high Gd loadings were obtained (between 1.8 and 14.5 × 104 Gd centres per NP). By tuning the cross-linker ionization degree and the nature of the polymer matrix it was possible to boost the r1 relaxivity per Gd centre up to 22-fold. The greatest gains in relaxivity were observed for nanogels for which the polymer matrix was constituted of chitosan and hyaluronan. Relaxivities per Gd centre as high as 100 s-1 mM-1 at 30 MHz can be reached, which highlighted the fact that molecular motion of the Gd chelate was effectively restricted and water access to the inner core of these nanogels was not limited.

Etoposide encapsulation in surface-modified poly(lactide-co-glycolide) nanoparticles strongly enhances glioma antitumor efficiency.

Etoposide (VP-16) is a hydrophobic anticancer agent inhibiting Topoisomerase II, commonly used in pediatric brain chemotherapeutic schemes as mildly toxic. Unfortunately, despite its appropriate solubilization in vehicle solvents, its poor bioavailability and limited passage of the blood-brain barrier concur to disappointing results requiring the development of new delivery system forms. In this study, etoposide formulated as a parenteral injectable solution (Teva®) was loaded into all-biocompatible poly(lactide-co-glycolide) (PLGA) or PLGA/P188-blended nanoparticles (size 110-130 nm) using a fully biocompatible nanoprecipitation technique. The presence of coprecipitated P188 on encapsulation efficacies and in vitro drug release was investigated. Drug encapsulation was determined using HPLC. Inflammatory response was checked by FACS analysis on human monocytes. Cytotoxic activity of the various simple (Teva®) or double (Teva®-loaded NPs) formulations was studied on the murine C6 and F98 cell lines. Obtained results suggest that, although noninflammatory neither nontoxic by themselves, the use of PLGA and PLGA/P188 nanoencapsulations over pre-existing etoposide formulation could induce a greatly improved cytotoxic activity. This approach demonstrated a promising perspective for parenteral delivery of VP16 and potential development of a therapeutic entity.

Hydrogels incorporating GdDOTA: towards highly efficient dual T1/T2 MRI contrast agents.

Do not tumble dry: Gadolinium-DOTA encapsulated into polysaccharide nanoparticles (GdDOTA NPs) exhibited high relaxivity (r(1) =101.7 s(-1) mM(-1) per Gd(3+) ion at 37 °C and 20 MHz). This high relaxation rate is due to efficient Gd loading, reduced tumbling of the Gd complex, and the hydrogel nature of the nanoparticles. The efficacy of the nanoparticles as a T(1)/T(2) dual-mode contrast agent was studied in C6 cells.

Serum albumin-alginate coated microspheres: role of the inner gel in binding and release of the KRFK peptide.

In continuation with our previous study using fluorescein-isothiocyanate (FITC)-Lys-Arg-Phe-Lys (KRFK) peptide, the aim of this work was to study the interaction of the unlabelled KRFK with calcium alginate gel microspheres coated with a serum albumin (HSA)-alginate membrane prepared using a transacylation method. Coated microspheres were prepared with two main sizes and two gel strengths. Control microspheres made of cross-linked alginate-HSA without calcium alginate gel were also prepared. A series of loading and release assays conducted with methylene blue showed the requirement of inner gel for binding the cationic molecule. Release experiments were performed in different media using unlabelled KRFK and coated microspheres. A plateau was reached within 1h, in contrast with the slow release of the FITC-peptide observed in our previous work. This discrepancy was attributed to modified properties of the labelled peptide. Adsorption assays of KRFK on coated microspheres were performed in the presence of growing concentrations of NaCl or imidazole. The ions were able to displace the peptide from the particles, which demonstrated ionic interactions, probably involving carboxylate groups of alginate. Adsorption isotherms showed that gel strength influenced affinity (4x10(5) L/mol or 8x10(5) L/mol for gelation with 5% or 20% CaCl(2), respectively). Binding site number doubled (from 2.6x10(-7) mol/mg to more than 5x10(-7) mol/mg) when microsphere size decreased from 450 microm to 100 microm. Binding sites were assumed to be located in the gel underneath the membrane.

Albumin-alginate-coated microspheres: resistance to steam sterilization and to lyophilization.

The paper describes the effect of different thermal treatments on the morphology and binding properties of particles prepared using a transacylation reaction between two biocompatible polymers, namely propylene glycol alginate and human serum albumin. Compared to control alginate gel microspheres, albumin-alginate covalent network offers a better resistance to the microspheres towards freezing, lyophilization and sterilization. The binding properties for methylene blue were not altered by the treatments. Moreover, stability in physiological environments opens interesting applications in biological and pharmaceutical fields.