LDLR-Mediated Targeting and Productive Uptake of siRNA-Peptide Ligand Conjugates In Vitro and In Vivo.

Small RNA molecules such as microRNA and small interfering RNA (siRNA) have become promising therapeutic agents because of their specificity and their potential to modulate gene expression. Any gene of interest can be potentially up- or down-regulated, making RNA-based technology the healthcare breakthrough of our era. However, the functional and specific delivery of siRNAs into tissues of interest and into the cytosol of target cells remains highly challenging, mainly due to the lack of efficient and selective delivery systems. Among the variety of carriers for siRNA delivery, peptides have become essential candidates because of their high selectivity, stability, and conjugation versatility. Here, we describe the development of molecules encompassing siRNAs against SOD1, conjugated to peptides that target the low-density lipoprotein receptor (LDLR), and their biological evaluation both in vitro and in vivo.

Preparation and In Vitro Validation of a Cucurbit[7]uril-Peptide Conjugate Targeting the LDL Receptor.

Here we report the coupling of a cyclic peptide (VH4127) targeting the low density lipoprotein (LDL) receptor (LDLR) noncompetitively to cucurbit[7]uril (CB[7]) to develop a new kind of drug delivery system (DDS), namely, CB[7]-VH4127, with maintained binding affinity to the LDLR. To evaluate the uptake potential of this bismacrocyclic compound, another conjugate was prepared comprising a high-affinity group for CB[7] (adamantyl(Ada)-amine) coupled to the fluorescent tracker Alexa680 (A680). The resulting A680-Ada·CB[7]-VH4127 supramolecular complex demonstrated conserved LDLR-binding potential and improved LDLR-mediated endocytosis and intracellular accumulation potential in LDLR-expressing cells. The combination of two technologies, namely, monofunctionalized CB[7] and the VH4127 LDLR-targeting peptide, opens new avenues in terms of targeting and intracellular delivery to LDLR-expressing tissues or tumors. The versatile transport capacity of CB[7], known to bind a large spectrum of bioactive or functional compounds, makes this new DDS suitable for a wide range of therapeutic or imaging applications.

Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos.

Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells-or organs that express the LDLR.

Anandamide-ceramide interactions in a membrane environment: Molecular dynamic simulations data.

Anandamide is a lipid neurotransmitter that interacts with various plasma membrane lipids. The data here consists of molecular dynamics simulations of anandamide, C18-ceramide and cholesterol performed in vacuo and within a hydrated palmitoyl-oleoyl-phosphatidylcholine (POPC)/cholesterol membrane. Several models of anandamide/cholesterol and anandamide/ceramide complexes are presented. The energy of interaction and the nature of the intermolecular forces involved in each of these complexes are detailed. The impact of water molecules hydrating the POPC/cholesterol membrane for the stability of the anandamide/cholesterol and anandamide/ceramide complexes is also analyzed. From a total number of 1920 water molecules stochatiscally merged with the lipid matrix, 48 were eventually redistributed around the polar head groups of the anandamide/ceramide complex, whereas only 15 reached with the anandamide/cholesterol complex. The interpretation of this dataset is presented in the accompanying article "Ceramide binding to anandamide increases its half-life and potentiates its cytotoxicity in human neuroblastoma cells" [1].

Ceramide binding to anandamide increases its half-life and potentiates its cytotoxicity in human neuroblastoma cells.

Anandamide (AEA) is a ubiquitous lipid that exerts neurotransmitter functions but also controls important biological functions such as proliferation, survival, or programmed cell death. The latter effects are also regulated by ceramide, a lipid enzymatically generated from sphingomyelin hydrolysis by sphingomyelinase. Ceramide has been shown to increase the cellular toxicity of AEA, but the mechanisms controlling this potentiating effect remained unclear. Here we have used a panel of in silico, physicochemical, biochemical and cellular approaches to study the crosstalk between AEA and ceramide apoptotic pathways. Molecular dynamics simulations indicated that AEA and ceramide could form a stable complex in phosphatidylcholine membranes. Consistent with these data, we showed that AEA can specifically insert into ceramide monolayers whereas it did not penetrate into sphingomyelin membranes. Then we have studied the effects of ceramide on AEA-induced toxicity of human neuroblastoma cells. In these experiments, the cells have been either naturally enriched in ceramide by neutral sphingomyelinase pre-incubation or treated with C2-ceramide, a biologically active ceramide analog. Both treatments significantly increased the cytotoxicity of AEA as assessed by the MTS mitochondrial toxicity assay. This effect was correlated with the concomitant accumulation of natural ceramide (or its synthetic analog) and AEA in the cells. A kinetic study of AEA hydrolysis showed that ceramide inhibited the fatty acid amino hydrolase (FAAH) activity in cell extracts. Taken together, these data suggested that ceramide binds to AEA, increases its half-life and potentiates its cytotoxicity. Overall, these mechanisms account for a functional cross-talk between AEA and ceramide apoptotic pathways.

Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier.

The blood-brain barrier (BBB) prevents the entry of many drugs into the brain and, thus, is a major obstacle in the treatment of CNS diseases. There is some evidence that the LDL receptor (LDLR) is expressed at the BBB and may participate in the transport of endogenous ligands from blood to brain, a process referred to as receptor-mediated transcytosis. We previously described a family of peptide vectors that were developed to target the LDLR. In the present study, in vitro BBB models that were derived from wild-type and LDLR-knockout animals (ldlr-/- ) were used to validate the specific LDLR-dependent transcytosis of LDL via a nondegradative route. We next showed that LDLR-targeting peptide vectors, whether in fusion or chemically conjugated to an Ab Fc fragment, promote binding to apical LDLR and transendothelial transfer of the Fc fragment across BBB monolayers via the same route as LDL. Finally, we demonstrated in vivo that LDLR significantly contributes to the brain uptake of vectorized Fc. We thus provide further evidence that LDLR is a relevant receptor for CNS drug delivery via receptor-mediated transcytosis and that the peptide vectors we developed have the potential to transport drugs, including proteins or Ab based, across the BBB.-Molino, Y., David, M., Varini, K., Jabès, F., Gaudin, N., Fortoul, A., Bakloul, K., Masse, M., Bernard, A., Drobecq, L., Lécorché, P., Temsamani, J., Jacquot, G., Khrestchatisky, M. Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier.

Optimization and in Vivo Validation of Peptide Vectors Targeting the LDL Receptor.

Active targeting and delivery to pathophysiological organs of interest is of paramount importance to increase specific accumulation of therapeutic drugs or imaging agents while avoiding systemic side effects. We recently developed a family of new peptide ligands of the human and rodent LDL receptor (LDLR), an attractive cell-surface receptor with high uptake activity and local enrichment in several normal or pathological tissues (Malcor et al., J. Med. Chem. 2012, 55 (5), 2227). Initial chemical optimization of the 15-mer, all natural amino acid compound 1/VH411 (DSGL[CMPRLRGC]cDPR) and structure-activity relationship (SAR) investigation led to the cyclic 8 amino acid analogue compound 22/VH445 ([cMPRLRGC]c) which specifically binds hLDLR with a KD of 76 nM and has an in vitro blood half-life of ∼3 h. Further introduction of non-natural amino acids led to the identification of compound 60/VH4106 ([(d)-"Pen"M"Thz"RLRGC]c), which showed the highest KD value of 9 nM. However, this latter analogue displayed the lowest in vitro blood half-life (∼1.9 h). In the present study, we designed a new set of peptide analogues, namely, VH4127 to VH4131, with further improved biological properties. Detailed analysis of the hLDLR-binding kinetics of previous and new analogues showed that the latter all displayed very high on-rates, in the 106 s-1.M-1 range, and off-rates varying from the low 10-2 s-1 to the 10-1 s-1 range. Furthermore, all these new analogues showed increased blood half-lives in vitro, reaching ∼7 and 10 h for VH4129 and VH4131, respectively. Interestingly, we demonstrate in cell-based assays using both VH445 and the most balanced optimized analogue VH4127 ([cM"Thz"RLRG"Pen"]c), showing a KD of 18 nM and a blood half-life of ∼4.3 h, that its higher on-rate correlated with a significant increase in both the extent of cell-surface binding to hLDLR and the endocytosis potential. Finally, intravenous injection of tritium-radiolabeled 3H-VH4127 in wild-type or ldlr -/- mice confirmed their active LDLR targeting in vivo. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential in vivo.

Mislocalization of the exitatory amino-acid transporters (EAATs) in human astrocytoma and non-astrocytoma cancer cells: effect of the cell confluence.

BACKGROUND: Astrocytomas are cancers of the brain in which high levels of extracellular glutamate plays a critical role in tumor growth and resistance to conventional treatments. This is due for part to a decrease in the activity of the glutamate transporters, i.e. the Excitatory Amino Acid Transporters or EAATs, in relation to their nuclear mislocalization in astrocytoma cells. Although non-astrocytoma cancers express EAATs, the localization of EAATs and the handling of L-glutamate in that case have not been investigated. METHODS: We looked at the cellular localization and activity of EAATs in human astrocytoma and non-astrocytoma cancer cells by immunofluorescence, cell fractionation and L-glutamate transport studies. RESULTS: We demonstrated that the nuclear mislocalization of EAATs was not restricted to astrocytoma and happened in all sub-confluent non-astrocytoma cancer cells we tested. In addition, we found that cell-cell contact caused the relocalization of EAATs from the nuclei to the plasma membrane in all human cancer cells tested, except astrocytoma. CONCLUSIONS: Taken together, our results demonstrated that the mislocalization of the EAATs and its associated altered handling of glutamate are not restricted to astrocytomas but were also found in human non-astrocytoma cancers. Importantly, we found that a cell contact-dependent signal caused the relocalization of EAATs at the plasma membrane at least in human non-astrocytoma cancer cells, resulting in the correction of the altered transport of glutamate in such cancer cells but not in astrocytoma.

The ribotoxin deoxynivalenol affects the viability and functions of glial cells.

Glial cells are responsible for maintaining brain homeostasis. Modification of the viability and functions of glial cells, including astrocytes and microglia, are associated with neuronal death and neurological diseases. Many toxins (heavy metals, pesticides, bacterial or viral toxins) are known to impact on brain cell viability and functions. Although recent publications suggest a potential link between environmental exposure of humans to mycotoxins and neurological diseases, data regarding the effects of fungal toxins on brain cells are scarce. In the present study, we looked at the impact of deoxynivalenol (DON), a fungal ribotoxin, on glial cells from animal and human origin. We found that DON decreased the viability of glial cells with a higher toxicity against microglial cells compared with astrocytes. In addition to cellular toxicity, DON affected key functions of glial cells. Thus, DON caused a biphasic effect on the neuroinflammatory response of microglia to lipopolysaccharide (LPS), while sublethal doses of DON increased the LPS-induced secretion of TNF-α and nitric oxide, toxic doses inhibited it. In addition to affecting microglial functions, sublethal doses of DON also suppressed the uptake of L-glutamate by astrocytes. This inhibition was associated with a modification of the expression of the glutamate transporters at the plasma membrane. Our results suggest that environmental ribotoxins such as DON could, at low doses, cause modifications of brain homeostasis and possibly participate in the etiology of neurological diseases in which alterations of the glia are involved.

The food-associated fungal neurotoxin ochratoxin A inhibits the absorption of glutamate by astrocytes through a decrease in cell surface expression of the excitatory amino-acid transporters GLAST and GLT-1.

The food-associated mycotoxin ochratoxin A (OTA) has been demonstrated to be deleterious to numerous cell types including brain cells. Although OTA has been proved to be toxic to astrocytes, no other investigation has been conducted on the impact of OTA on astrocytic functions. In the present study, we evaluated the effect of OTA on one of the major astrocytic functions, i.e. the reabsorption of extracellular glutamate. We found that OTA suppressed glutamate absorption by rat cortical astrocytes with a half inhibitory concentration of 1.3 and 10.1 microM in the absence and presence of fetal calf serum. Although OTA inhibits glutamine synthetase activity, this effect was not involved in OTA-mediated alteration of glutamate absorption since decrease in enzyme activity only occurred at high cytotoxic concentrations of toxin (100 microM). Similarly, alterations in the expression of the excitatory amino-acid transporters were not involved since OTA failed to modify total expression level of GLAST and GLT-1. We found that inhibition of glutamate absorption by OTA was due to a decrease in the expression of GLAST and GLT-1 at the cell surface. We propose that, in addition to being directly toxic to neurons and astrocytes, OTA could also cause the death of brain cells through inhibition of glutamate uptake by astrocytes, leading to the accumulation of extracellular glutamate and ultimately to excitotoxicity.