The use of liposomes functionalized with the NFL-TBS.40-63 peptide as a targeting agent to cross the in vitro blood-brain barrier and target glioblastoma cells.

Glioblastoma is the most common and aggressive brain tumor. Current treatments do not allow to cure the patients. This is partly due to the blood-brain barrier (BBB), which limits the delivery of drugs to the pathological site. To overcome this, we developed liposomes functionalized with a neurofilament-derived peptide, NFL-TBS.40-63 (NFL), known for its highly selective targeting of glioblastoma cells. First, in vitro BBB model was developed to check whether the NFL can also promote barrier crossing in addition to its active targeting capacity. Permeability experiments showed that the NFL peptide was able to cross the BBB. Moreover, when the BBB was in a pathological situation, i.e., an in vitro blood-brain tumor barrier (BBTB), the passage of the NFL peptide was greater while maintaining its glioblastoma targeting capacity. When the NFL peptide was associated to liposomes, it enhanced their ability to be internalized into glioblastoma cells after passage through the BBTB, compared to liposomes without NFL. The cellular uptake of liposomes was limited in the endothelial cell monolayer in comparison to the glioblastoma one. These data indicated that the NFL peptide is a promising cell-penetrating peptide tool when combined with drug delivery systems for the treatment of glioblastoma.

Enhanced and preferential internalization of lipid nanocapsules into human glioblastoma cells: effect of a surface-functionalizing NFL peptide.

Increasing intracellular drug concentration using nanocarriers can be a potential strategy to improve efficacy against glioblastoma (GBM). Here, the fluorescent-labelled NFL-TBS·40-63 peptide (fluoNFL) concentration on a lipid nanocapsule (LNC) was studied to enhance nanovector internalization into human GBM cells. LNC surface-functionalization with various fluoNFL concentrations was performed by adsorption. LNC size and surface charge altered gradually with increasing peptide concentration, but their complement protein consumption remained low. Desorption of fluoNFL from the LNC surface was found to be slow. Furthermore, it was observed that the rate and extent of LNC internalization in the U87MG human glioblastoma cells were dependent on the surface-functionalizing fluoNFL concentration. In addition, we showed that the uptake of fluoNFL-functionalized LNCs was preferential towards U87MG cells compared to healthy human astrocytes. The fluoNFL-functionalized LNC internalization into the U87MG cells was energy-dependent and occurred possibly by macropinocytosis and clathrin-mediated and caveolin-mediated endocytosis. A new ferrocifen-type molecule (FcTriOH), as a potent anticancer candidate, was then encapsulated in the LNCs and the functionalization improved its in vitro efficacy compared to other tested formulations against U87MG cells. In the preliminary study, on subcutaneous human GBM tumor model in nude mice, a significant reduction of relative tumor volume was observed at one week after the second intravenous injection with FcTriOH-loaded LNCs. These results showed that enhancing NFL peptide concentration on the LNC surface is a promising approach for increased and preferential nanocarrier internalization into human GBM cells, and the FcTriOH-loaded LNCs are a promising therapy approach for GBM.

The Neurofilament-Derived Peptide NFL-TBS.40-63 Targets Neural Stem Cells and Affects Their Properties.

UNLABELLED: Targeting neural stem cells (NSCs) in the adult brain represents a promising approach for developing new regenerative strategies, because these cells can proliferate, self-renew, and differentiate into new neurons, astrocytes, and oligodendrocytes. Previous work showed that the NFL-TBS.40-63 peptide, corresponding to the sequence of a tubulin-binding site on neurofilaments, can target glioblastoma cells, where it disrupts their microtubules and inhibits their proliferation. We show that this peptide targets NSCs in vitro and in vivo when injected into the cerebrospinal fluid. Although neurosphere formation was not altered by the peptide, the NSC self-renewal capacity and proliferation were reduced and were associated with increased adhesion and differentiation. These results indicate that the NFL-TBS.40-63 peptide represents a new molecular tool to target NSCs to develop new strategies for regenerative medicine and the treatment of brain tumors. SIGNIFICANCE: In the present study, the NFL-TBS.40-63 peptide targeted neural stem cells in vitro when isolated from the subventricular zone and in vivo when injected into the cerebrospinal fluid present in the lateral ventricle. The in vitro formation of neurospheres was not altered by the peptide; however, at a high concentration of the peptide, the neural stem cell (NSC) self-renewal capacity and proliferation were reduced and associated with increased adhesion and differentiation. These results indicate that the NFL-TBS.40-63 peptide represents a new molecular tool to target NSCs to develop new strategies for regenerative medicine and the treatment of brain tumors.

The NFL-TBS.40-63 anti-glioblastoma peptide disrupts microtubule and mitochondrial networks in the T98G glioma cell line.

Despite aggressive therapies, including combinations of surgery, radiotherapy and chemotherapy, glioblastoma remains a highly aggressive brain cancer with the worst prognosis of any central nervous system disease. We have previously identified a neurofilament-derived cell-penetrating peptide, NFL-TBS.40-63, that specifically enters by endocytosis in glioblastoma cells, where it induces microtubule destruction and inhibits cell proliferation. Here, we explore the impact of NFL-TBS.40-63 peptide on the mitochondrial network and its functions by using global cell respiration, quantitative PCR analysis of the main actors directing mitochondrial biogenesis, western blot analysis of the oxidative phosphorylation (OXPHOS) subunits and confocal microscopy. We show that the internalized peptide disturbs mitochondrial and microtubule networks, interferes with mitochondrial dynamics and induces a rapid depletion of global cell respiration. This effect may be related to reduced expression of the NRF-1 transcription factor and of specific miRNAs, which may impact mitochondrial biogenesis, in regard to default mitochondrial mobility.

Review on intermediate filaments of the nervous system and their pathological alterations.

Intermediate filaments (IFs) of the nervous system, including neurofilaments, α-internexin, glial fibrillary acidic protein, synemin, nestin, peripherin and vimentin, are finely expressed following elaborated cell, tissue and developmental specific patterns. A common characteristic of several neurodegenerative diseases is the abnormal accumulation of neuronal IFs in cell bodies or along the axon, often associated with impairment of the axonal transport and degeneration of neurons. In this review, we also present several perturbations of IF metabolism and organization associated with neurodegenerative disorders. Such modifications could represent strong markers of neuronal damages. Moreover, recent data suggest that IFs represent potential biomarkers to determine the disease progression or the differential stages of a neuronal disorder. Finally, recent investigations on IF expression and function in cancer provide evidence that they may be useful as markers, or targets of brain tumours, especially high-grade glioma. A better knowledge of the molecular mechanisms of IF alterations, combined to neuroimaging, is essential to improve diagnosis and therapeutic strategies of such neurodegenerative diseases and glioma.

The NFL-TBS.40-63 anti-glioblastoma peptide enters selectively in glioma cells by endocytosis.

Glioblastoma are the most frequent and aggressive tumour of the nervous system despite surgical resection associated with chemotherapy and radiotherapy. Recently, we showed that the NFL-TBS.40-63 peptide corresponding to the sequence of a tubulin-binding site of neurofilaments, enters selectively in glioblastoma cells where it blocks microtubule polymerization, inhibits their proliferation, and reduces tumour development in rats bearing glioblastoma (Bocquet et al., 2009; Berges et al., 2012a). Here, we characterized the molecular mechanism responsible for the uptake of NFL-TBS.40-63 peptide by glioblastoma cells. Unlike other cell penetrating peptides (CPPs), which use a balance between endocytosis and direct translocation, the NFL-TBS.40-63 peptide is unable to translocate directly through the membrane when incubated with giant plasma membrane vesicles. Then, using a panel of markers and inhibitors, flow cytometry and confocal microscopy investigations showed that the uptake occurs mainly through endocytosis. Moreover, glycosaminoglycans and αVß3 integrins are not involved in the NFL-TBS.40-63 peptide recognition and internalization by glioblastoma cells. Finally, the signalling of tyrosine kinase receptors is involved in the peptide uptake, especially via EGFR overexpressed in tumour cells, indicating that the uptake of NFL-TBS.40-63 peptide by glioblastoma cells is related to their abnormally high proliferative activity.