Versatile electrostatically assembled polymeric siRNA nanovectors: Can they overcome the limits of siRNA tumor delivery?

The application of small interfering RNA (siRNA) cancer therapeutics is limited by several extra- and intracellular barriers including the presence of ribonucleases that degrade siRNA, the premature clearance, the impermeability of the cell membrane, or the difficulty to escape endo-lysosomal degradation. Therefore, several delivery systems have emerged to overcome these limitations and to successfully deliver siRNA to the tumor site. This review is focused on polymer-based siRNA nanovectors which exploit the negative charge of siRNA, representing a major challenge for siRNA delivery, to their advantage by loading siRNA via electrostatic assembly. These nanovectors are easy to prepare and to adapt for an optimal gene silencing efficiency. The ability of electrostatically assembled polymeric siRNA nanovectors (EPSN) to improve the half-life of siRNA, to favor the specificity of the delivery and the accumulation in tumor and to enhance the cellular uptake and endosomal escape for an efficient siRNA delivery will be discussed. Finally, the influence of the versatility of the structure of these nanovectors on the protein down-regulation will be evaluated.

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

OBJECTIVE: 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. METHODS: Lipid-based encapsulation systems (LNC) were prepared by the PIT method. Their physicochemical properties were followed over 6 months by dynamic light scattering and zeta potential measurements, and in parallel, the potential degradation of the active ingredient was monitored by HPLC. The stability of the capsules in a cosmetic gel was studied by spectrofluorimetry and rheology measurements. Sensory analysis was used to determine the influence of the presence of capsules in the gel on the consumer's experience. RESULTS: LNC encapsulating Omegalight® were prepared on a laboratory scale and then on a semi-pilot scale. Their hydrodynamic diameters are around 230 nm. The concentration of Omegalight® in the capsules reaches about 84% w/w, which corresponds to 42% of active ingredient. LNC can be dispersed without degradation at concentrations of up to 20% w/w in a hydrogel without modification of the physicochemical or sensory properties of the gel. CONCLUSION: Lipid-based capsules (LNC), an encapsulation system useful for the epidermal delivery of hydrophobic compounds, were adapted to the encapsulation of a commercial lightening agent. The encapsulation permits the dispersion in a stable manner of a very high concentration of a hydrophobic active molecule in a hydrogel while maintaining the physicochemical and sensory properties of the gel.

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.

Recent advances in theranostic nanocarriers of doxorubicin based on iron oxide and gold nanoparticles.

Hybrid (organic/inorganic) nanoparticles emerged as a simple solution to build "theranostic" systems. Due to their physical properties, superparamagnetic iron oxide nanoparticles (SPIONs) and plasmonic gold nanoparticles (Au-NPs) are extensively studied as a part of diagnostic and therapeutic strategies in cancer treatments. They can be used as agents for in vitro or in vivo imaging, for magnetic drug targeting and/or thermal therapy. Their functionalization with organic shells enhances their potential performance in tumor targeting and drug delivery. The advances in such hybrid nanocarriers are well illustrated with the example of the anticancer drug doxorubicin (DOX). The aim of this review is to give a multidisciplinary overview of such smart nanosystems loaded with DOX, based on examples taken from recent publications. From a physico-chemical point of view, we discuss the choices for the strategies for loading DOX and the consequences on drug release. From a biological point of view, we analyze the in vitro and in vivo assays concerning tumor imaging, targeted drug delivery and anticancer efficiency. Future opportunities and challenges are also addressed.

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.

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.

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.

The development of stable aqueous suspensions of PEGylated SPIONs for biomedical applications.

We report here the development of stable aqueous suspensions of biocompatible superparamagnetic iron oxide nanoparticles (SPIONs). These so-called ferrofluids are useful in a large spectrum of modern biomedical applications, including novel diagnostic tools and targeted therapeutics. In order to provide prolonged circulation times for the nanoparticles in vivo, the initial iron oxide nanoparticles were coated with a biocompatible polymer poly(ethylene glycol) (PEG). To permit covalent bonding of PEG to the SPION surface, the latter was functionalized with a coupling agent, 3-aminopropyltrimethoxysilane (APS). This novel method of SPION PEGylation has been reproduced in numerous independent preparations. At each preparation step, particular attention was paid to determine the physico-chemical characteristics of the samples using a number of analytical techniques such as atomic absorption, Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy, transmission electron microscopy (TEM), photon correlation spectroscopy (PCS, used for hydrodynamic diameter and zeta potential measurements) and magnetization measurements. The results confirm that aqueous suspensions of PEGylated SPIONs are stabilized by steric hindrance over a wide pH range between pH 4 and 10. Furthermore, the fact that the nanoparticle surface is nearly neutral is in agreement with immunological stealthiness expected for the future biomedical applications in vivo.

Comparative study of doxorubicin-loaded poly(lactide-co-glycolide) nanoparticles prepared by single and double emulsion methods.

This study describes how the control of doxorubicin (DOX) polarity allows to encapsulate it inside poly(lactide-co-glycolide) (PLGA) nanoparticles formulated either by a single oil-in-water (O/W) or a double water-in-oil-in-water (W/O/W) emulsification method (SE and DE, respectively). DOX is commercially available as a water soluble hydrochloride salt, which is useful for DE. The main difficulty related to DE approach is that the low affinity of hydrophilic drugs to the polymer limits entrapment efficiency. Compared to DE method, SE protocol is easier and should provide an additional gain in entrapment efficiency. To be encapsulated by SE technique, DOX should be used in a more lipophilic molecular form. We evaluated the lipophilicity of DOX in terms of apparent partition coefficient (P) and modulated it by adjusting the pH of the aqueous phase. The highest P values were obtained at pH ranging from 8.6 to 9, i. e. between two DOX pK(a) values (8.2 and 9.6). The conditions favorable for the drug lipophilicity were then used to formulate DOX-loaded PLGA nanoparticles by SE method. DOX encapsulation efficiency as well as release profiles were evaluated for these nanoparticles and compared to those with nanoparticles formulated by DE. Our results indicate that the encapsulation of DOX in nanoparticles formulated by SE provides an increased drug entrapment efficiency and decreases the burst effect.