Multifunctional hybrid nanoplatform based on Fe3O4@Ag NPs for nitric oxide delivery: development, characterization, therapeutic efficacy, and hemocompatibility.

The combination of Fe3O4@Ag superparamagnetic hybrid nanoparticles and nitric oxide (NO) represents an innovative strategy for a localized NO delivery with a simultaneous antibacterial and antitumoral actions. Here, we report the design of Fe3O4@Ag hybrid nanoparticles, coated with a modified and nitrosated chitosan polymer, able to release NO in a biological medium. After their synthesis, physicochemical characterization confirmed the obtention of small NO-functionalized superparamagnetic Fe3O4@Ag NPs. Antibacterial assays demonstrated enhanced effects compared to control. Bacteriostatic effect against Gram-positive strains and bactericidal effect against E. coli were demonstrated. Moreover, NO-functionalized Fe3O4@Ag NPs demonstrated improved ability to reduce cancer cells viability and less cytotoxicity against non-tumoral cells compared to Fe3O4@Ag NPs. These effects were associated to the ability of these NPs act simultaneous as cytotoxic (necrosis inductors) and cytostatic compounds inducing S-phase cell cycle arrest. NPs also demonstrated low hemolysis ratio (<10%) at ideal work range, evidencing their potential for biomedical applications. Targeted and hemocompatible nitric oxide-releasing multi-functional hybrid nanoparticles for antitumor and antimicrobial applications.

The Impact of Multiple Functional Layers in the Structure of Magnetic Nanoparticles and Their Influence on Albumin Interaction.

In nanomedicine, hybrid nanomaterials stand out for providing new insights in both the diagnosis and treatment of several diseases. Once administered, engineered nanoparticles (NPs) interact with biological molecules, and the nature of this interaction might directly interfere with the biological fate and action of the NPs. In this work, we synthesized a hybrid magnetic nanostructure, with antibacterial and antitumoral potential applications, composed of a magnetite core covered by silver NPs, and coated with a modified chitosan polymer. As magnetite NPs readily oxidize to maghemite, we investigated the structural properties of the NPs after addition of the two successive layers using Mössbauer spectroscopy. Then, the structural characteristics of the NPs were correlated to their interaction with albumin, the major blood protein, to evidence the consequences of its binding on NP properties and protein retention. Thermodynamic parameters of the NPs-albumin interaction were determined. We observed that the more stable NPs (coated with modified chitosan) present a lower affinity for albumin in comparison to pure magnetite and magnetite/silver hybrid NPs. Surface properties were key players at the NP-biological interface. To the best of our knowledge, this is the first study that demonstrates a correlation between the structural properties of complex hybrid NPs and their interaction with albumin.

Evaluation of biocompatible stabilised gelled soya bean oil nanoparticles as new hydrophobic reservoirs.

Based on the organogel concept, in which an oil is trapped in a network of low-molecular-mass organic gelator fibres creating a gel, a formulation of gelled soya bean oil nanoparticles was evaluated for its capacity to form biocompatible hydrophobic reservoirs. The aqueous dispersions of nanoparticles were prepared by hot emulsification (T° > Tgel) and cooling at room temperature in the presence of polyethyleneimine (PEI). The dispersions were stabilised by the electrostatic interactions between the positively charged amino groups of the PEI and the negatively charged carboxylates of the gelator fibres present at the surface of the particles. The aqueous dispersions were highly stable (several months) and the gelled particles were able to entrap a hydrophobic fluorescent model molecule (Nile red), allowing testing in cells. The gelled oil nanoparticles were found to be biocompatible with the tested cells (keratinocytes) and had the ability to become rapidly internalised. Thus, organogel-based nanoparticles are a promising hydrophobic drug delivery system.

Design of surface ligands for blood compatible gold nanoparticles: Effect of charge and binding energy.

Gold nanoparticle (AuNP) interaction with the blood compartment as a function of their charge and the binding energy of their surface ligand was explored. Citrate, polyallylamine and cysteamine stabilized AuNP along with dihydrolipoic acid and polyethylene glycol capped AuNP were synthesized and fully characterized. Their interactions with model proteins (human albumin and human fibrinogen) were studied. Complexes formed between AuNP and protein revealed several behaviors ranging from corona formation to aggregation. Protein fluorescence quenching as a function of temperature and AuNP concentration allowed the determination of the thermodynamic parameters describing these interactions. The hemolysis induced by AuNP was also probed: an increasing or a decreasing of hemolysis ratio induced by AuNP was observed as of function of protein corona formation. Taken together, our results drew up a composite sketch of an ideal surface ligand for blood compatible AuNP. This capping agent should be strongly bound to the gold core by one or more thiol groups and it must confer a negative charge to the particles.

Dextran-covered pH-sensitive oily core nanocapsules produced by interfacial Reversible Addition-Fragmentation chain transfer miniemulsion polymerization.

Aiming to prepare oily core pH-sensitive nanocapsules (NCs) for anticancer drugs delivery, the use of a dextran-based transurf (DexN3-τCTAγ) as both stabilizer and macromolecular chain transfer agent in methyl methacrylate/2-(diethylamino)ethyl methacrylate (MMA/DEAEMA) miniemulsion copolymerization was investigated. NCs of about 195 nm with an oily-core of Miglyol 810 (M810) and a dextran coverage covalently linked to the poly(MMA-co-DEAEMA) intern shell have been obtained. Compared to the non-sensitive PMMA-based NCs (prepared in a similar way), these novel objects were shown to swell in acidic media and to trigger Coumarin 1 release in physiological relevant pH range. As a starting point of NCs biological effects, cytotoxicity and NCs-proteins interactions studies were performed with both PMMA and poly(MMA-co-DEAEMA)-based NCs. Finally, free azide functions from dextran-based coverage were successfully exploited to attach fluorescent model dyes to NCs surface. The overall results suggest that this novel NCs platform could be potentially used as drug nanocarriers for intravenous injection.

Nitric oxide-eluting scaffolds and their interaction with smooth muscle cells in vitro.

Fabrication of scaffolds loaded with nitric oxide (NO) donors (S-nitrosoglutathione, GSNO, and isosorbide mononitrate, ISMN) with suitable cell compatibility and optimized properties for tissue-engineering applications is reported using "in situ" technique. Based on FDA-approved polymer, solvent and dosage forms, this gentle process allowed the incorporation of the GSNO labile drug into scaffolds made of either poly(lactide-co-glycolide) (PLGA) or PLGA/poly(ɛ-caprolactone) (PCL) blend. During scaffolds manufacturing process including washing cycles, NO donors were leached from scaffolds. However, GSNO and ISMN concentrations in the last washing medium (10(-7) M and 10(-4) M, respectively) were in the range of cell suitability for tissue engineering. Further morphological analyses indicated that smoother surfaces with fewer but bigger pores (compatible with cell penetration and ingrowth) were obtained with PLGA in comparison with PLGA/PCL scaffolds. Among all tested matrices, only unloaded PLGA and GSNO-loaded PLGA/PCL exhibited intermediate cell anchorage, with mitochondrial activity close to the control and an increase in protein content, a prognostic for scaffold cell colonization, defining them as promising candidates. Deeper analyses of these two scaffolds looking at intracellular redox balance through reactive oxygen species production, glutathione, S-nitrosothiols, and nitrite ions content exhibited GSNO-loaded PLGA/PCL as the best of all tested 3D scaffolds for tissue engineering.

PLGA in situ implants formed by phase inversion: critical physicochemical parameters to modulate drug release.

In situ forming implants (ISI) based on phase separation by solvent exchange represent an attractive alternative to conventional preformed implants and microparticles for parenteral applications. They are indeed easier to manufacture and their administration does not require surgery, therefore improving patient compliance. They consist of polymeric solutions precipitating at the site of injection and thus forming a drug eluting depot. Drug release from ISI is typically divided into three phases: burst during precipitation of the depot, diffusion of drug through the polymeric matrix and finally drug release by system degradation. This review gives a comprehensive overview on (i) the theoretical bases of these three phases, (ii) the parameters influencing them and (iii) the remaining drawbacks which have to be addressed to enlarge their commercial opportunities. Indeed, although some of them are already commercialized, ISI still suffer from limitations: mainly lack of reproducibility in depot shape, burst during solidification and potential toxicity. Nevertheless, depending on the targeted therapeutic application, these shortcomings may be transformed into advantages. As a result, keys are given in order to tailor these formulations in view of the desired application so that ISI could gain further clinical importance in the following years.

Are in situ formulations the keys for the therapeutic future of S-nitrosothiols?

S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) were formulated into in situ forming implants (ISI) and microparticles (ISM) using PLGA and either N-methyl-2-pyrrolidone (NMP) or triacetin. Physicochemical characterization was carried out, including the study of matrix structure and degradation. A strong correlation between drug hydrophobicity and the in vitro release profiles was observed: whatever the formulation, GSNO and SNAP were completely released after ca. 1 day and 1 week, respectively. Then, selected formulations (i.e., SNAP-loaded NMP formulations) demonstrated the ability to sustain the vasodilation effect of SNAP, as shown by monitoring the arterial pressure (telemetry) of Wistar rats after subcutaneous injection. Both ISI and ISM injections resulted in a 3-fold extended decrease in pulse arterial pressure compared with the unloaded drug, without significant decrease in the mean arterial pressure. Hence, the results emphasize the suitability of these formulations as drug delivery systems for S-nitrosothiols, widening their therapeutic potential.

Development of tripartite polyion micelles for efficient peptide delivery into dendritic cells without altering their plasticity.

For many years, a great deal of interest has been focusing on the optimization of peptide presentation by dendritic cells (DCs) using peptide-encapsulated particles, in order to enhance the immune response. Nowadays, DCs are also known to be involved in peripheral tolerance, inducing anergy or regulatory T lymphocytes. To preserve the plasticity of DCs, we formulated non-cytotoxic pH-sensitive polyion complex micelles based on an original tripartite association of polymethacrylic acid-b-polyethylene oxide, poly-L-lysine and fluorescent-peptide: OVAFITC peptide, as a model drug. We demonstrated that the OVAFITC peptide was successfully entrapped into the micelles, released into DC endosomes thanks to the pH-sensitivity property of the micelles, and efficiently loaded onto MHC class II molecules. The phenotype as well as the cytokinic secretion profile of the mature and immature DCs loaded with peptide-encapsulated micelles was unaltered by the tripartite polyion micelles. The efficient loading of the peptide by immature and mature DCs was shown by the in vitro proliferation of OVA-specific transgenic T cells. Therefore, the present results show that the tripartite polyion complex micelles can be used as efficient peptide vectors immunogically inert for ex vivo DCs engineering without modifying their intrinsic immune plasticity.

pH-sensitive double-hydrophilic block copolymer micelles for biological applications.

In the recent years, double-hydrophilic block copolymer (DHBC) micelles have appeared as potential vectors for pharmaceutical applications due to their simple preparation method in aqueous solvent. The present study aims at underscoring the strategy for the choice of the partners in the formulation of DHBC micelles presenting a good stability in physiological conditions (pH 7.4, 0.15 mol/L NaCl) and a pH-sensitivity allowing their disassembly at pH 5. Using light scattering and Laser-Doppler electrophoresis, micelles of polymethacrylic acid-b-polyethylene oxide complexing either poly-l-lysine (PLL) or an oligochitosan were characterised. Whatever the polyamine counter-polyion considered, the micelles were perfectly formed for an amine/methacrylic acid molar charge ratio of one. They were characterised by a hydrodynamic diameter of 28 nm for PLL and 60 nm for oligochitosan and by a neutral zeta potential. The stability study as a function of the pH and of the ionic strength revealed different behaviours. Oligochitosan micelles were stable until pH 7 and unstable at 0.15 mol/L NaCl. On the contrary, PLL micelles were stable in physiological conditions and disassembled at pH 5. As a conclusion, the choice of the partners to formulate double-hydrophilic block copolymer based-micelles is strategic in order to obtain well-adapted vectors applied to the pharmaceutical field.

The control of dendritic cell maturation by pH-sensitive polyion complex micelles.

Double-hydrophilic block copolymer micelles were designed as vectors for ex vivo dendritic cell engineering to improve the delivery of therapeutic molecules in such immune cells. Polymethacrylic acid-b-polyethylene oxide (PMAA(2100)-b-POE(5000))/poly-L-lysine micelles were optimised and showed a hydrodynamic diameter of 30 nm with a peculiar core organised with hydrogen bonds as well as hydrophobic domains. The micelles proved high stability in physiological conditions (pH and ionic strength) and were also able to disassemble under acidic conditions mimicking acidic endolysosomes. The efficient endocytosis of the optimised micelles tested on bone marrow-derived dendritic cells was monitored by fluorescence-activated cell sorting and microscopy analysis. Finally, the micelle biocompatibility permitted a complete control of the dendritic cell-maturation process widening the therapeutical potential of such engineered dendritic cells for cancer vaccines as well as for immunomodulation in autoimmune diseases.