Katel Hervé-Aubert

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.

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.

Synthesis and in vitro evaluation of fluorescent and magnetic nanoparticles functionalized with a cell penetrating peptide for cancer theranosis

We synthesized rationally designed multifunctional nanoparticles (NPs) composed of a superparamagnetic iron oxide nanoparticle (SPION) core, cyanine fluorescent dye emitting in far red, polyethylene glycol (PEG5000) coating, and the membranotropic peptide gH625, from the cell-penetrating peptides (CPP) family. The peptide sequence was enriched with an additional cysteine so it can be involved as a reactive moiety in a certain orientation- and sequence-specific coupling of the CPP to the PEG shell of the NPs. Our data indicate that the presence of approximately 23 peptide molecules per SPION coated with approximately 137 PEG chains minimally changes the overall NP characteristics. The final CPP-capped NP hydrodynamic diameter was 98nm, the polydispersity index was 0.192, and the zeta potential was 4.08mV. The in vitro evaluation, performed using an original technique fluorescence confocal spectral imaging, showed that after a short incubation duration (maximum 30min), SPIONs-PEG-CPP uptake was 3-fold higher than that for SPIONs-PEG. The CPP also drives the subcellular distribution of a higher NP fraction towards low polarity cytosolic locations. Therefore, the major cellular uptake mechanism for the peptide-conjugated NPs should be endocytosis. Enhancement/acceleration of this mechanism by gH625 appears promising because of potential applications of SPIONs-PEG-gH625 as a multifunctional nanoplatform for cancer theranosis involving magnetic resonance imaging, optical imaging in far red, drug delivery, and hyperthermia.

Covalent conjugation of cysteine-engineered scFv to PEGylated magnetic nanoprobes for immunotargeting of breast cancer cells

In the present study, we describe the synthesis and characterization of new generation of cancer-targeting magnetic nanoprobes: superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylene glycol (PEG) shell functionalized with recombinant anti-HER2 single chain fragment variable (scFv) of Trastuzumab antibody. An anti-HER2 scFv with terminal cysteine (scFv 4D5-Cys) has been rationally engineered in order to favor its orientation- and site-directed covalent conjugation to the polymeric surface of PEGylated SPIONs. Optimization of scFv and nanoparticles production allowed to obtain well-characterized SPIONs-PEG–scFv nanoparticles carrying ∼7 fragments per nanoparticle, having a hydrodynamic diameter of ca. 86 nm and nearly neutral surface. The nanoprobes-scFv capability to recognize the HER2 protein has been confirmed by enzyme-linked immunosorbent assay (ELISA). Compared to non-targeted PEGylated SPIONs, the SPIONs–PEG–scFv nanoprobes showed an enhanced binding to HER2-overexpressing cells (SK-BR3) in vitro as it was shown by immunofluorescence. Finally, ICP-AES measurements shown that in 1 hour the uptake of SPIONs–PEG–scFv in HER2-overexpressing cells is 2.1 times greater than non-targeted PEGylated SPIONs. Therefore, both due to their physico-chemical characteristics and the immunotargeting of HER2-positive breast cancer cells, the SPIONs–PEG–scFv appear as promising nanoplatforms for future applications in theranostic treatment of cancers.

Use of experimental design methodology for the development of new magnetic siRNA nanovectors (MSN)

Short interfering RNAs (siRNAs) can downregulate the synthesis of proteins and thus be used to treat certain diseases where the protein synthesis is upregulated, such as cancer. The challenge is to deliver siRNAs in the target cell as they are rapidly degraded by nucleases and have difficulties to cross the cellular membranes. Superparamagnetic iron oxide nanoparticles (SPIONs) are widely studied as platforms for smart biocompatible nanosystems which can be used for magnetic drug targeting and magnetic resonance imaging. The aim of this work was to combine siRNAs, SPIONs, and chitosan, to develop new magnetic siRNA nanovectors suitable for systemic administration. In a first time, the one factor at a time (OFAT) methodology was used to adjust different formulation parameters and to test the feasibility of such a formulation. In a second time, design of experiment (DOE) methodology was used to analyze the influence of these formulation parameters on the physicochemical characteristics hydrodynamic diameter (DH) and ζ-potential. Finally, four MSNs suitable for systemic administration could be identified using the OFAT method. The DOE method showed a significant effect of CR and [NaNO3] on the DH and a significant effect of MR and [siRNA] on the ζ-potential of the nanocarriers.

Folic acid-capped PEGylated magnetic nanoparticles enter cancer cells mostly via clathrin-dependent endocytosis

BACKGROUND This work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA. METHODS SFP-FA were characterized by DLS, zetametry and fluorescence spectroscopy. The SFP-FA uptake in cancer cells was monitored using fluorescence-based methods like fluorescence-assisted cell sorting, CLSM with single-photon and two-photon excitation. The SFP-FA endocytosis was also analyzed with electron microscopy approaches: TEM, HAADF-STEM and EELS. RESULTS The SFP-FA have zeta potential below -6mW and stable hydrodynamic diameter close to 100nm in aqueous suspensions of pH range from 5 to 8. They contain ca. 109 PEG-FA, 480 PEG-OCH3 and 22-27 fluorophore molecules per SPION. The fluorophores protected under the PEG shell allows a reliable detection of intracellular NPs. SFP-FA readily enter into all the cancer cell lines studied and accumulate in lysosomes, mostly via clathrin-dependent endocytosis, whatever the FR status on the cells. CONCLUSIONS The present study highlights the advantages of rational design of nanosystems as well as the possible involvement of direct molecular interactions of PEG and FA with cellular membranes, not limited to FA-FR recognition, in the mechanisms of their endocytosis. GENERAL SIGNIFICANCE Composition, magnetic and optical properties of the SFP-FA as well their ability to enter cancer cells are promising for their applications in cancer theranosis. Combination of complementary analytical approaches is relevant to understand the nanoparticles behavior in suspension and in contact with cells.

siRNA delivery system based on magnetic nanovectors: Characterization and stability evaluation

Abstract Gene therapy and particularly small interfering RNA (siRNA) is a promising therapeutic method for treatment of various human diseases, especially cancer. However the lack of an ideal delivery system limits its clinical applications. Effective anticancer drug development represents the key for translation of research advances into medicines. Previously we reported, the optimization of magnetic siRNA nanovectors (MSN) formulation based on superparamagnetic iron oxide nanoparticles (SPION) and chitosan for systemic administration. This work aimed at using rational design to further optimize and develop MSN. Therefore, formulated MSN were first purified, then their physical and chemical properties were studied mainly through capillary electrophoresis. 95% of siRNA was found enclosed within the purified MSN (pMSN). pMSN showed colloidal stability at pH 7.4, effective protection of siRNA against ribonuclease degradation up to 24 hours and few siRNA release (less than 10%) at pH 7.4. These findings push toward further evaluation studies in vitro and/or in vivo, indicating the appropriateness of pMSN for cancer theranostics. Graphical abstract Figure. No Caption available.

Stealth magnetic nanocarriers of siRNA as platform for breast cancer theranostics

The endogenous mechanism of RNA interference is more and more used in research to obtain specific down-regulation of gene expression in diseases such as breast cancer. Currently, despite the new fields of study open up by RNA interference, the rapid degradation of siRNA by nucleases and their negative charges prevent them from crossing cell membranes. To overcome these limitations, superparamagnetic iron oxide nanoparticles (SPIONs) represent a promising alternative for nucleic acid delivery. Previously, we reported the magnetic siRNA nanovectors (MSN) formulation using electrostatic assembly of (1) SPIONs, also able to act as contrast agents for magnetic resonance imaging (MRI), (2) siRNA and (3) chitosan aiming at their protection and enhancing their transfection efficacy. However, these nanoparticles displayed low stability in biological suspensions and inefficient transfection of active siRNA. This work aimed at upgrading MSN to Stealth MSN (S-MSN) by adding a polyethylene glycol coating to ensure colloidal stability and stealth properties. Furthermore, another polymer (poly-L-arginine) was added for efficient siRNA transfection and the quantitative composition of the formulation was adapted for biological purposes. Results showed that S-MSN provide high siRNA complexation and protection against enzymatic degradation. Green fluorescent protein (GFP) specific down-regulation on MDA-MB231/GFP cells was comparable to that of commercially available reagents, without observable cytotoxicity. According to our works, S-MSN appears as an effective formulation for in vitro siRNA specific delivery.

Formulation and in vitro evaluation of a siRNA delivery nanosystem decorated with gH625 peptide for triple negative breast cancer theranosis

Graphical abstract Figure. No caption available. ABSTRACT The development of an efficient small interfering RNA (siRNA) delivery system has held scientists interest since the discovery of the RNA interference mechanism (RNAi). This strategy gives hope for the treatment of many severe diseases. Herein, we developed hybrid nanovectors able to deliver siRNA to triple negative breast cancer cells. The nanovectors are based on PEGylated superparamagnetic iron oxide nanoparticles (SPION) functionalized with gH625 peptide, chitosan and poly‐l‐arginine. Every component has a key role and specific function: SPION is the core scaffolding the nanovector; PEG participates in the colloidal stability and the immune stealthiness; gH625 peptide promotes the nanovector internalization into cancer cells; cationic polymers provide the siRNA protection and favor siRNA endosomal escape and delivery to cytosol. The formulation was optimized by varying the amount of each compound. The efficacy of the siRNA retention and protection were investigated in the presence of high concentration of serum. Optimized nanovectors show a high uptake by MDA‐MB‐231 cells. The resulting down regulation of GFP expression was 73 ± 3% with our nanovector compared to 59 ± 8% obtained with the siRNA‐Oligofectamine™ complex in the same conditions.

Impact of Site-Specific Conjugation of ScFv to Multifunctional Nanomedicines Using Second Generation Maleimide

Biocompatible multifunctional nanomedicines (NMs) are known to be an attractive platform for targeted anticancer theranosis. However, these nanomedicines are of interest only if they efficiently target diseased cells and accumulate in tumors. Here we report the synthesis of a new generation of immunotargeted nanomedicines composed of a superparamagnetic iron oxide nanoparticle (SPION) core, polyethylene glycol coating and the anti-HER2 single chain fragment variable (scFv) of Trastuzumab antibody. We developed two novel bioengineered scFv carrying two cysteines located (i) at the end (4D5.1-cys2) or (ii) at the beginning (4D5.2-cys2) of its hexahistidine tag. The scFv bioconjugation was controlled via heterobifunctional linkers including a second generation maleimide (SGM). Our data indicated that the insertion of cysteines at the beginning of the hexahistidine tag was allowed to obtain nearly 2-fold conjugation efficiency (13 scFv/NP) compared to NMs using classical maleimide. As a result, the NMs-4D5.2 built using the optimal 4D5-cys2 and linkers equipped with SGM showed the enhanced recognition of HER2 in an ELISA format and on the surface of SK-BR-3 breast cancer cells in vitro. Their stability in serum was also significantly improved compared to the NMs-4D5. Our results showed the fundamental importance of the controlled ligand conjugation in the perspective of rational design of NMs with tailored physicochemical and biological properties.