Emilie Munnier

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.

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.

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.

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.

Qualitative and Quantitative Study of the Potential of Lipid Nanocapsules of One Hundred Twenty Nanometers for the Topical Administration of Hydrophobic Molecules

In this study, we evaluated the potential of lipid nanocapsules (LNC) of 120 nm as drug nanocarriers to treat skin diseases. As a model molecule, we encapsulated the fluorescent dye curcumin, which also is an antioxidant. Curcumin-loaded LNC showed interesting antioxidant properties and a low toxicity on human skin cells. The penetration of curcumin in the skin was determined by 2 complementary methods: high performance liquid chromatography was used to measure total curcumin accumulation in the skin, whereas fluorescence confocal spectral imaging of skin sections showed that curcumin preferentially accumulates in the stratum corneum and the viable epidermis. These results confirm that LNC of a size above 100 nm can vectorize hydrophobic compounds to the keratinocytes without transdermal delivery. They also demonstrate the interest of combining 2 analytical methods when studying the skin penetration of nanovectorized molecules.

Toward multifunctional hybrid platforms for tissue engineering based on chitosan(PEO) nanofibers functionalized by bare laser-synthesized Au and Si nanoparticles

Exhibiting a variety of unique optical, structural and physicochemical properties, laser-synthesized nanomaterials have become increasingly popular during recent years in a variety of biomedical, catalytic, photovoltaic and other applications. Here, we explore the use of bare laser-synthesized gold and silicon nanoparticles (AuNPs and SiNPs) as additives to functionalize electrospun chitosan(PEO) nanofibers and then assess the potential of such hybrid structures as multifunctional platforms for tissue engineering. We demonstrate that bare AuNPs and SiNPs can be easily grafted on the surface of the chitosan(PEO) nanofibers without any interference, via electrostatic interaction between a strong negative surface charge of NPs and the polycationic surface of the fibers. We also show that the nanofibers functionalized with nanoparticles can affect the morphology and physico-chemical characteristics of the resulting nanostructures. As an example, the functionalization of nanofibers by SiNPs led to quite different thicknesses of fibers (386 ± 80 nm and 632 ± 170 nm), suggesting a potential improvement of fibre surface reactivity. Finally, biological toxicity of the nanofibers was assessed through preliminary viability tests conducted on HaCaT cells. After 24 h of incubation time, no adverse effects were observed confirming satisfactory biocompatibilty of the hybrid nanofiber structures. The proposed concept promises exciting perspectives in the development of innovative multifunctional scaffolds structures gathering new properties for tissue engineering.

Lipid-based submicron capsules as a strategy to include high concentrations of a hydrophobic lightening agent in a hydrogel

This study aimed at increasing the concentration of a hydrophobic lightening agent, Omegalight®, in a hydrophilic cosmetic product by means of encapsulation in lipid‐based submicron capsules. The core of these capsules is entirely made of the commercial lightening agent.

Bare laser-synthesized Si nanoparticles as functional elements for chitosan nanofiber-based tissue engineering platforms

Methods of femtosecond laser ablation were used to fabricate bare (ligand-free) silicon (Si) nanoparticles in deionized water. The nanoparticles were round in shape, crystalline, free of any impurities, and water-dissolvable, while the dissolution rate depended on the concentration of oxygen defects in their composition. The nanoparticles were then eletrospun with chitosan to form nanoparticle decorated nanofibrous matrices. We found that the functionalization of nanofibers by the nanoparticles can affect the morphology and physico-chemical characteristics of resulting nanostructures. In particular, the presence of Si nanoparticles led to the reduction of fibers thickness, suggesting a potential improvement of fiber’s surface reactivity. We also observed the improvement of thermal stability of hybrid nanofibers. We believe that the incorporated Si nanoparticles can serve as functional elements to improve characteristics of chitosan-based matrices for cellular growth, as well as to enable novel imaging or therapeutic functionalities for tissue engineering applications.

Confocal Raman Spectroscopic Imaging for In Vitro Monitoring of Active Ingredient Penetration and Distribution in Reconstructed Human Epidermis Model

Topically applied active cosmetic ingredients (ACI) or active pharmaceutical ingredients (API) efficacy is directly related to their efficiency of penetration in the skin. In vitro reconstructed human epidermis surrogate models offer in vivo like skin samples for transdermal studies. Using Delipidol®, an ACI currently used in the cosmetics industry, the capabilities to deliver accurate distribution maps and penetration profiles of this molecule by means of confocal Raman spectroscopic imaging have been demonstrated. Using a non-negative constrained least squares (NCLS) approach, contribution of specific molecules can be estimated at each point of spectral maps in order to deliver semi-quantitative heat maps representing the ACI levels in the different skin layers. The concentration profiles obtained are approximately single exponential for all 3 time points evaluated, with a consistent decay constant, which is independent of the sublayer structure. Notably, however, there is no significant penetration into the lower basal layers until a critical concentration is built up, after 3 hours. Combination of Raman confocal imaging with spectral unmixing methods such as NCLS is demonstrated to be a relevant approach for in vitro biological evaluation of cosmetic and pharmaceutical active ingredients and could easily be implemented as a screening tool for industrial use.