Martin Soucé

Molecular composition of iron oxide nanoparticles, precursors for magnetic drug targeting, as characterized by confocal Raman microspectroscopy

The chemical and structural properties of ferrite-based nanoparticles, precursors for magnetic drug targeting, have been studied by Raman confocal multispectral imaging. The nanoparticles were synthesised as aqueous magnetic fluids by co-precipitation of ferrous and ferric salts. Dehydrated particles corresponding to co-precipitation (CP) and oxidation (OX) steps of the magnetic fluid preparation have been compared in order to establish oxidation-related Raman features. These are discussed in correlation with the spectra of bulk iron oxides (magnetite, maghemite and hematite) recorded under the same experimental conditions. Considering a risk of laser-induced conversion of magnetite into hematite, this reaction was studied as a function of laser power and exposure to oxygen. Under hematite-free conditions, the Raman data indicated that nanoparticles consisted of magnetite and maghemite, and no oxyhydroxide species were detected. The relative maghemite/magnetite spectral contributions were quantified via fitting of their characteristic bands with Lorentzian profiles. Another quality parameter, contamination of the samples with carbon-related species, was assessed via a broad Raman band at 1580 cm(-1). The optimised Raman parameters permitted assessment of the homogeneity and stability of the solid phase of prepared magnetic fluids using chemical imaging by Raman multispectral mapping. These data were statistically averaged over each image and over six independently prepared lots of each of the CP and OX nanoparticles. The reproducibility of oxidation rates of the particles was satisfactory: the maghemite spectral fraction varied from 27.8 +/- 3.6% for the CP to 43.5 +/- 5.6% for the OX samples. These values were used to speculate about the layered structure of isolated particles. Our data were in agreement with a model with maghemite core and magnetite nucleus. The overall oxidation state of the particles remained nearly unchanged for at least one month.

Novel method of doxorubicin–SPION reversible association for magnetic drug targeting

A new method of reversible association of doxorubicin (DOX) to superparamagnetic iron oxide nanoparticles (SPION) is developed for magnetically targeted chemotherapy. The efficacy of this approach is evaluated in terms of drug loading, delivery kinetics and cytotoxicity in vitro. Aqueous suspensions of SPION (ferrofluids) were prepared by coprecipitation of ferric and ferrous chlorides in alkaline medium followed by surface oxidation by ferric nitrate and surface treatment with citrate ions. The ferrofluids were loaded with DOX using a pre-formed DOX-Fe(2+) complex. The resulting drug loading was as high as 14% (w/w). This value exceeds the maximal loading known from literature up today. The release of DOX from the nanoparticles is strongly pH-dependent: at pH 7.4 the amount of drug released attains a plateau of approximately 85% after 1h, whereas at pH 4.0 the release is almost immediate. At both pH, the released drug is iron-free. The in vitro cytotoxicity of the DOX-loaded SPION on the MCF-7 breast cancer cell line is similar to that of DOX in solution or even higher, at low-drug concentrations. The present study demonstrates the potential of the novel method of pH-sensitive DOX-SPION association to design novel magnetic nanovectors for chemotherapy.

Magnetic nanocarriers of doxorubicin coated with poly(ethylene glycol) and folic acid: relation between coating structure, surface properties, colloidal stability, and cancer cell targeting

We report the efficient one-step synthesis and detailed physicochemical evaluation of novel biocompatible nanosystems useful for cancer therapeutics and diagnostics (theranostics). These systems are the superparamagnetic iron oxide nanoparticles (SPIONs) carrying the anticancer drug doxorubicin and coated with the covalently bonded biocompatible polymer poly(ethylene glycol) (PEG), native and modified with the biological cancer targeting ligand folic acid (PEG-FA). These multifunctional nanoparticles (SPION-DOX-PEG-FA) are designed to rationally combine multilevel mechanisms of cancer cell targeting (magnetic and biological), bimodal cancer cell imaging (by means of MRI and fluorescence), and bimodal cancer treatment (by targeted drug delivery and by hyperthermia effect). Nevertheless, for these concepts to work together, the choice of ingredients and particle structure are critically important. Therefore, in the present work, a detailed physicochemical characterization of the organic coating of the hybrid nanoparticles is performed by several surface-specific instrumental methods, including surface-enhanced Raman scattering (SERS) spectroscopy, X-ray photoelectron spectrometry (XPS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). We demonstrate that the anticancer drug doxorubicin is attached to the iron oxide surface and buried under the polymer layers, while folic acid is located on the extreme surface of the organic coating. Interestingly, the moderate presence of folic acid on the particle surface does not increase the particle surface potential, while it is sufficient to increase the particle uptake by MCF-7 cancer cells. All of these original results contribute to the better understanding of the structure-activity relationship for hybrid biocompatible nanosystems and are encouraging for the applications in cancer theranostics.

A pharmaceutical study of doxorubicin-loaded PEGylated nanoparticles for magnetic drug targeting

One of the new strategies to improve cancer chemotherapy is based on new drug delivery systems, like the polyethylene glycol-coated superparamagnetic iron oxide nanoparticles (PEG-SPION, thereafter called PS). In this study, PS are loaded with doxorubicin (DOX) anticancer drug, using a pre-formed DOX-Fe(2+) complex reversible at lower pH of tumour tissues and cancer cells. The DOX loaded PS (DLPS, 3% w/w DOX/iron oxide) present a hydrodynamic size around 60nm and a zeta potential near zero at physiological pH, both parameters being favourable for increased colloidal stability in biological media and decreased elimination by the immune system. At physiological pH of 7.4, 60% of the loaded drug is gradually released from the DLPS in ∼2h. The intracellular release and distribution of DOX is followed by means of confocal spectral imaging (CSI) of the drug fluorescence. The in vitro cytotoxicity of the DLPS on MCF-7 breast cancer cells is equivalent to that of a DOX solution. The reversible association of DOX to the SPION surface and the role of polymer coating on the drug loading/release are discussed, both being critical for the design of novel stealth magnetic nanovectors for chemotherapy.

Poly(ethylene glycol)-stabilized silver nanoparticles for bioanalytical applications of SERS spectroscopy

The present work depicts the efficient one-step synthesis and detailed evaluation of stable aqueous colloids of silver nanoparticles (NPs) coated with poly(ethylene glycol) (PEG) covalently attached to their surface. Due to steric repulsion between polymer-modified surfaces, the stability of the nanoparticle suspension was preserved even at high ionic strength (0.1 M NaCl). At the same time, the PEG coating remains sufficiently permeable to allow surface-enhanced Raman scattering (SERS) from micromolar concentrations of small molecules such as the anticancer drug mitoxantrone (MTX). The enhancement efficiency of the hot spot-free Ag-PEG was compared to that of citrate-stabilized Ag colloids used after pre-aggregation. The potential of the polymer-stabilized colloids developed in this study is discussed in terms of bioanalytical applications of SERS spectroscopy.

Pegylated magnetic nanocarriers for doxorubicin delivery: A quantitative determination of stealthiness in vitro and in vivo

The aim of this work was to elucidate the impact of polyethylene glycol (PEG) polymeric coating on the in vitro and in vivo stealthiness of magnetic nanocarriers loaded or not with the anticancer drug doxorubicin. The comparison was made between aqueous suspensions of superparamagnetic iron oxide nanoparticles (SPIONs) stabilized by either citrate ions (C-SPIONs) or PEG(5000) (P-SPIONs), the latter being loaded or not with doxorubicin via the formation of a DOX-Fe(2+) complex (DLP-SPIONs). After determination of their relevant physico-chemical properties (size and surface charge), nanoparticle (NP) stealthiness was studied in vitro (ability to activate the complement system and uptake by monocytes and macrophage-like cells) and in vivo in mice (blood half-life; t(1/2), and biodistribution in main clearance organs). These aspects were quantitatively assessed by atomic absorption spectrometry (AAS). Complement activation dramatically decreased for sterically stabilized P-SPIONs and DLP-SPIONs in comparison with C-SPIONs stabilized by charge repulsion. Monocyte and macrophage uptake was also largely reduced for pegylated formulations loaded or not with doxorubicin. The t(1/2) in blood for P-SPIONs was estimated to be 76 ± 6 min, with an elimination mainly directed to liver and spleen. Thanks to their small size (<80 nm) and a neutral hydrophilic polymer-extended surface, P-SPIONs exhibit prolonged blood circulation and thus potentially an increased level in tumor delivery suitable for magnetic drug targeting applications.

Design strategies of hybrid metallic nanoparticles for theragnostic applications

Metallic nanoparticles (MNPs) such as iron oxide and gold nanoparticles are interesting platforms to build theragnostic nanocarriers which combine both therapeutic and diagnostic functions within a single nanostructure. Nevertheless, their surface must be functionalized to be suitable for in vivo applications. Surface functionalization also provides binding sites for targeting ligands, and for drug loading. This review focuses on the materials and surface chemistry used to build hybrid nanocarriers that are inorganic cores functionalized with organic materials. The surface state of the MNPs largely depends on their synthesis routes, and dictates the strategies used for functionalization. Two main strategies can be found in the literature: the design of core-shell nanosystems, or embedding nanoparticles in organic materials. Emerging tendencies such as the use of clusters or alternative coating materials are also described. To present both hydrophilic and lipophilic nanosystems, we chose the doxorubicin anticancer agent as an example, as the molecule presents an affinity for both types of materials.

Novel alginate-based nanocarriers as a strategy to include high concentrations of hydrophobic compounds in hydrogels for topical application

The cutaneous penetration of hydrophobic active molecules is of foremost concern in the dermatology and cosmetic formulation fields. The poor solubility in water of those molecules limits their use in hydrophilic forms such as gels, which are favored by patients with chronic skin disease. The aim of this work is to design a novel nanocarrier of hydrophobic active molecules and to determine its potential as an ingredient of a topical form. The nanocarrier consists of an oily core surrounded by a protective shell of alginate, a natural polysaccharide isolated from brown algae. These calcium alginate-based nanocarriers (CaANCs) were prepared at room temperature and without the use of organic solvent by an accelerated nanoemulsification-polymer crosslinking method. The size (hydrodynamic diameter ~200 nm) and surface charge (zeta potential ~ - 30 mV) of the CaANCs are both compatible with their application on skin. CaANCs loaded with a fluorescent label were stable in model hydrophilic galenic forms under different storage conditions. Curcumin was encapsulated in CaANCs with an efficiency of ~95%, fully retaining its antioxidant activity. The application of the curcumin-loaded CaANCs on excised human skin led to a significant accumulation of the active molecules in the upper layers of the skin, asserting the potential of these nanocarriers in active pharmaceutical and cosmetic ingredients topical delivery.

On the interaction of alginate-based core-shell nanocarriers with keratinocytes in vitro.

Calcium alginate nanocarriers (CaANCs) were developed as a potential tool for delivery of hydrophobic active molecules such as pharmaceutical and cosmetic active ingredients. In this study, we focused on interactions between CaANCs and keratinocytes in culture and examined toxicity, internalization and drug release. Prior to cellular interactions, cryogenic transmission electron microscopy images showed that CaANCs appear as regular, spherical and dense particles, giving evidence of the surface gelation of CaANCs. Their size, around 200nm, was stable under tested conditions (temperature, culture media, presence of serum and presence of encapsulated dye), and their toxicity on keratinocytes was very low. Flow cytometry assays showed that CaANCs are internalized into keratinocytes by endocytosis with a predominant implication of the caveolae-mediated route. Förster resonance energy transfer (FRET) demonstrated that after a 2h contact, the release of CaANC contents in the cytoplasm of keratinocytes was almost complete. The endocytosis of CaANCs by a lysosome-free pathway, and the rapid release of their contents inside keratinocytes, will allow vectorized molecules to fully exhibit their pharmacological or cosmetic activity.

Colloidal stability and thermo-responsive properties of iron oxide nanoparticles coated with polymers: advantages of Pluronic® F68-PEG mixture.

Superparamagnetic iron oxide nanoparticles (SPIONs) are recognized to be an attractive platform for developing novel drug delivery approaches and thus several types of functionalized magnetic nanocarriers based on SPIONs have been synthesized and studied. The coating of the metal oxide surface was achieved in a one-pot synthesis with biocompatible polyethylene glycol (PEG) and thermo-responsive modified Pluronic® F68. The resulting thermo-responsive magnetic nanocarriers can incorporate water insoluble drugs into their hydrophobic compartment and later release them in a temperature dependent manner. Here we report novel magnetic nanocarriers with significant improvements regarding the colloidal stability and critical temperature obtained by mixing various molar ratios of hydrophilic PEG with thermo-responsive Pluronic® F68 bearing different end group functionalities. Various methods have been employed to characterize the magnetic nanocarriers, such as photon correlation spectroscopy (DLS), atomic absorption, FT-IR spectroscopy, and surface-enhanced Raman scattering. The transition temperature that determines changes in the conformation of the block copolymer chain was studied by DLS as a function of temperature. Moreover, the drug loading properties of SPION-(F68-OMe)-(F68-FA) and SPION-PEG-F68-FA were analyzed with a hydrophobic fluorescent dye, DID oil. The behavior of the encapsulated DID into the nanocarrier shell was studied as a function of temperature via fluorescence spectroscopy. These results offer original insights into the enhanced colloidal stability and thermo-sensitive properties of the novel synthesized magnetic nanocarriers.