Investigating the functionalization of liposomes with NFL-TBS. 40-63 peptide as a promising drug delivery system.

The NFL-peptide was discovered almost 20 years ago, and its targeting properties were assessed alone or in combination with lipid nanocapsules (LNC), magnetic porous silicon nanorods, or gold nanoparticles. Results highlighted a better targeting of cancer cells, in particular glioblastoma and pancreas cancer. Considering the large use of liposomes (LPs) as an hydrophilic drug delivery system, this study explored the possibility to functionalize liposomes with three different sequences of NFL-peptides: native (NFL-peptide), biotinylated (BIOT-NFL) and coupled to fluorescein (FAM-NFL). Dynamic Light Scattering (DLS) complemented by cryo-electron microscopy (CEM) showed a peculiar ultrastructural arrangement between NFL-peptides and liposomes. Based on this architectural interaction, we investigated the biological contribution of these peptides in LPs-DiD glioblastoma cellular uptake. Flow cytometry complemented by confocal microscopy experiments demonstrated a consequent and systematic increased uptake of LPs-DiD into F98 cells when their surface was decorated with NFL-peptides. The intra-cellular distribution of these liposomes via an organelle tracker indicated the presence of LPs-DiD in lysosomes after 4 h. Based on the properties of this NFL-peptide, we showed in this work the crucial role of NFL peptide as an effective and promising actor to potentiate nanoparticles entry in glioblastoma cell lines.

Investigation on the self-assembly of the NFL-TBS.40-63 peptide and its interaction with gold nanoparticles as a delivery agent for glioblastoma.

NFL-TBS.40-63 peptide is a recently discovered peptide derived from the light neurofilament chain (NFL). In this study, we demonstrated that the Biotinylated-NFL-peptide (BIOT-NFL) can spontaneously self-assemble into well-organized nanofibers (approximately 5 nm width and several micrometers in length) in several solutions, whereas the typical self-assembly was not systematically observed from other peptides with or without coupling. The critical aggregation concentration that allows the BIOT-NFL-peptide to aggregate and auto associate was determined at 10-4 mol/L by surface tension measurements. X-ray scattering of BIOT-NFL-peptide also demonstrated its beta-sheet structure that can facilitate the intermolecular interactions involved in the self-assembly process. The possible disassembly of self-assembled BIOT-NFL-peptide-nanofibers was examined via a dialysis membrane study. We further investigated the interaction between nanofibers formed by BIOT-NFL-peptide and gold nanoparticles. Interestingly, a strong interaction was demonstrated between these nanoparticles and BIOT-NFL-peptide resulted in the formation of BIOT-NFL-peptide-nanofibers grandly decorated by gold nanoparticles. Finally, we investigated the internalization of gold nanoparticles coupled with BIOT-NFL-nanofibers into F98 rat glioblastoma cells, which was increased compared to the non-coupled control gold nanoparticles. All these results indicate that this peptide could be a promising therapeutic agent for targeted delivery.

Modeling nigrostriatal degeneration in organotypic cultures, a new ex vivo model of Parkinson's disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder afflicting 2% of the population older than 65 years worldwide. Recently, brain organotypic slices have been used to model neurodegenerative disorders, including PD. They conserve brain three-dimensional architecture, synaptic connectivity and its microenvironment. This model has allowed researchers a simple and rapid method to observe cellular interactions and mechanisms. In the present study, we developed an organotypic PD model from rat brains that includes all the areas involved in the nigrostriatal pathway in a single slice preparation, without using neurotoxins to induce the dopaminergic lesion. The mechanical transection of the nigrostriatal pathway obtained during slice preparation induced PD-like histopathology. Progressive nigrostriatal degeneration was monitored combining innovative approaches, such as diffusion tensor magnetic resonance imaging (DT-RMI) to follow fiber degeneration and mass spectrometry to quantify striatal dopamine content, together with bright-field and fluorescence microscopy imaging. A substantia nigra dopaminergic cell number decrease was observed by immunohistochemistry against rat tyrosine hydroxylase (TH) reaching 80% after 2 days in culture associated with a 30% decrease of striatal TH-positive fiber density, a 15% loss of striatal dopamine content quantified by mass spectrometry and a 70% reduction of nigrostriatal fiber fractional anisotropy quantified by DT-RMI. In addition, a significant decline of medium spiny neuron density was observed from days 7 to 16. These sagittal organotypic slices could be used to study the early stage of PD, namely dopaminergic degeneration, and the late stage of the pathology with dopaminergic and GABAergic neuron loss. This novel model might improve the understanding of PD and may represent a promising tool to refine the evaluation of new therapeutic approaches.

Perfluorocarbon-loaded lipid nanocapsules as oxygen sensors for tumor tissue pO2 assessment.

The assessment of tumor oxygenation is a crucial factor in cancer therapy and may be carried out using fluorine MRI once fluorine probes have been distributed within the tumor. However, the deposit of those highly fluorinated compounds often jeopardizes anatomical image quality and requires emulsification of the probes. Due to the high density and the high lipophilicity of perfluorocarbons, nanoemulsion of these molecules usually requires high-energy processes. In the present work, we discuss the synthesis and the physico-chemical characterization of perfluorocarbon nanocapsules using a low-energy phase-inversion process. The nanocapsules were tested on a mouse tumor brain model to assess oxygenation.

Development and characterization of immuno-nanocarriers targeting the cancer stem cell marker AC133.

In the context of targeted therapy, we addressed the possibility of developing a drug delivery nanocarrier capable to specifically reach cancer cells that express the most prominent marker associated with cancer stem cell (CSC) phenotype, AC133. For this purpose, 100nm lipid nanocapsules (LNCs) were functionalized with a monoclonal antibody (mAb) directed against AC133 according to two distinct methods: firstly, post-insertion within 100nm LNCs of a lipid poly(ethylene glycol) functionalized with reactive-sulfhydryl maleimide groups (DSPE-PEG(2000)-maleimide) followed by thiolated mAb coupling, and, secondly, creation of a thiolated lipo-immunoglobulin between DSPE-PEG(2000)-maleimide and AC133, then post-inserted within LNCs. Due to the reduced number of purification steps, lower amounts of DSPE-PEG(2000)-maleimide that were necessary as well as lower number of free maleimide functions present onto the surface of immuno-LNC, the second method was found to be more appropriate. Thus, 126nm AC133-LNC with a zeta potential of -22mV while keeping a narrow distribution were developed. Use of the IgG1κ isotype control-immunoglobulins produced similar control IgG1-LNCs. Micro-Bradford colorimetric assay indicated a fixation of about 40 immunoglobulins per LNC. Use of human Caco-2 cells that constitutively express AC133 (Caco-2-AC133(high)) allowed addressing the behavior of the newly functionalized immuno-LNCs. siRNA knockown strategy permitted to obtain Caco-2-AC133(low) for comparison. Immunofluorescence-combined flow cytometry analysis demonstrated that the epitope-recognition function of AC133 antibody was preserved when present on immuno-LNCs. Although grafting of immunoglobulins onto the surface of LNCs repressed their internalization within Caco-2 cells as evaluated by flow cytometry, AC133-specific cellular binding was obtained with AC133-LNC as assessed by computer-assisted fluorescence microscopy. In conclusion, interest of AC133-LNCs as niche carriers is discussed toward the development of CSC targeted chemo- or radio-nanomedicines.

Design of reversibly core cross-linked micelles sensitive to reductive environment.

Azido-functional amphiphilic macromolecules based on a biodegradable aliphatic polyester (poly-ε-caprolactone, PCL) and a bioeliminable hydrophilic poly(ethylene oxide) (PEO) block have been used in order to build micellar drug delivery systems. Such azido groups being able to react by alkyne-azide 1,3 Huisgens cycloaddition (a click reaction) have been used further in order to cross-link the micelles via redox-sensitive disulfide bridges. This reversible cross-linking allows to prevent micelle dissociation at high dilution upon injection and to trigger their dissociation in more reductive environment, such as the cytosol. Copolymers having three different architectures, i.e. able to cross-link either the core or the shell of core-shell-corona system have been used to investigate their micellization, cross-linking and cross-linking reversibility. The stealthiness of these micelles cross-linked in the hydrophobic segment has also been studied in vitro.