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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.

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.

The search for reliable human blood-brain barrier (BBB) models represents a challenge for the development/testing of strategies aiming to enhance brain delivery of drugs. Human-induced pluripotent stem cells (hiPSCs) have raised hopes in the development of predictive BBB models. Differentiating strategies are thus required to generate endothelial cells (ECs), a major component of the BBB. Several hiPSC-based protocols have reported the generation of in vitro models with significant differences in barrier properties. We studied in depth the properties of iPSCs byproducts from two protocols that have been established to yield these in vitro barrier models. Our analysis/study reveals that iPSCs derivatives endowed with EC features yield high permeability models while the cells that exhibit outstanding barrier properties show principally epithelial cell-like (EpC) features. We found that models containing EpC-like cells express tight junction proteins, transporters/efflux pumps and display a high functional tightness with very low permeability, which are features commonly shared between BBB and epithelial barriers. Our study demonstrates that hiPSC-based BBB models need extensive characterization beforehand and that a reliable human BBB model containing EC-like cells and displaying low permeability is still needed., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: The 5XFAD model of Alzheimer's disease (AD) bearing five familial mutations of Alzheimer's disease on human APP and PSEN1 transgenes shows deposits of amyloid-β peptide (Aβ) as early as 2 months, while deficits in long-term memory can be detected at 4 months using the highly sensitive olfactory-dependent tests that we previously reported., Objective: Given that detecting early dysfunctions in AD prior to overt pathology is of major interest in the field, we sought to detect memory deficits at earlier stages of the disease in 3-month-old male 5XFAD mice., Methods: To this end, we used the Helico Maze, a behavioral task that was recently developed and patented. This device allows deeper analysis of learning and subcategories of hippocampal-dependent long-term memory using olfactory cues., Results: Eight male 5XFAD and 6 male wild-type (WT: C57Bl6 background) mice of 3 months of age were tested in the Helico Maze. The results demonstrated, for the first time, a starting deficit of pure reference long-term memory. Interestingly, memory impairment was clearly correlated with Aβ deposits in the hippocampus. While we also found significant differences in astrogliosis between 5XFAD and WT mice, this was not correlated with memory abilities., Conclusion: Our results underline the efficiency of this new olfactory-dependent behavioral task, which is easy to use, with a small cohort of mice. Using the Helico Maze may open new avenues to validate the efficacy of treatments that target early events related to the amyloid-dependent pathway of the disease and AD progression.

Neurotensin (NT) acts as a primary neurotransmitter and neuromodulator in the CNS and has been involved in a number of CNS pathologies including epilepsy. NT mediates its central and peripheral effects by interacting with the NTSR1, NTSR2, and Sort1/NTSR3 receptor subtypes. To date, little is known about the precise expression of the NT receptors in brain neural cells and their regulation in pathology. In the present work, we studied the cellular distribution of the NTSR2 protein in the rat hippocampus and questioned whether its expression was modulated in conditions of neuroinflammation using a model of temporal lobe epilepsy induced by pilocarpine. This model is characterized by a rapid and intense inflammatory reaction with reactive gliosis in the hippocampus. We show that NTSR2 protein is expressed in hippocampal astrocytes and its expression increases together with astrocyte reactivity following induction of status epilepticus. NTSR2 immunoreactivity is also increased in astrocytes proximal to blood vessels and their end-feet, and in endothelial cells. Proinflammatory factors such as IL1β and LPS induced NTSR2 mRNA and protein in cultured astroglial cells. Antagonizing NTSR2 with SR142948A decreased NTSR2 expression as well as astroglial reactivity. Together, our results suggest that NTSR2 is implicated in astroglial and gliovascular inflammation and that targeting the NTSR2 receptor may open new avenues in the regulation of neuroinflammation in CNS diseases., (© 2021 Wiley Periodicals LLC.)

Despite clinical advances in diagnosis and treatment, pancreatic ductal adenocarcinoma (PDAC) remains the third leading cause of cancer death, and is still associated with poor prognosis and dismal survival rates. Identifying novel PDAC-targeted tools to tackle these unmet clinical needs is thus an urgent requirement. Here we use a peptide conjugate that specifically targets PDAC through low-density lipoprotein receptor (LDLR). We demonstrate by using near-infrared fluorescence imaging the potential of this conjugate to specifically detect and discriminate primary PDAC from healthy organs including pancreas and from benign mass-forming chronic pancreatitis, as well as detect metastatic pancreatic cancer cells in healthy liver. This work paves the way towards clinical applications in which safe LDLR-targeting peptide conjugate promotes tumor-specific delivery of imaging and/or therapeutic agents, thereby leading to substantial improvements of the PDAC patient's outcome., (© 2021. The Author(s).)

Targeted delivery to specific tissues and subcellular compartments is of paramount importance to optimize therapeutic or diagnostic interventions while minimizing side-effects. Using recently identified LDL receptor (LDLR) -targeting small synthetic peptide-vectors conjugated to model cargos of different nature and size, we investigated in LDLR-expressing cells the impact of vector-cargo molecular engineering and coupling valency, as well as the cellular exposure duration on their target engagement and intracellular trafficking and delivery profiles. All vector-cargo conjugates evaluated were found to be delivered to late compartments together with the natural ligand LDL, although to varying extents and with different kinetics. Partial recycling together with the LDLR was also consistently observed. Under continuous cellular exposure, the extent of intracellular vector-cargo delivery primarily relies on their endosomal unloading potential. In this condition, the highest intracellular delivery potential was observed with a monovalent conjugate displaying a rather high LDLR dissociation rate. On the contrary, under transient cellular exposure followed by chase, low dissociation-rate bivalent conjugates revealed a higher intracellular delivery potential than the monovalent conjugate. This was shown to rely on their ability to undergo multiple endocytosis-recycling rounds, with limited release in the ligand-free medium. The absence of reciprocal competition with the natural ligand LDL on their respective intracellular trafficking was also demonstrated, which is essential in terms of potential safety liabilities. These results demonstrate that not only molecular engineering of new therapeutic conjugates of interest, but also the cellular exposure mode used during in vitro evaluations are critical to anticipate and optimize their delivery potential., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The blood-brain barrier (BBB) regulates the traffic of molecules into the central nervous system (CNS) and also limits the drug delivery. Due to their flexible properties, liposomes are an attractive tool to deliver drugs across the BBB. We previously characterized gH625, a peptide derived from Herpes simplex virus 1. The present study investigates the efficiency of liposomes functionalized on their surface with gH625 to promote the brain uptake of neuroprotective peptide PACAP (pituitary adenylate cyclase-activating polypeptide). Using a rat in vitro BBB model, we showed that the liposomes preparations were non-toxic for the endothelial cells, as assessed by analysis of tight junction protein ZO1 organization and barrier integrity. Next, we found that gH625 improves the transfer of liposomes across endothelial cell monolayers, resulting in both low cellular uptake and increased transport of PACAP. Finally, in vivo results demonstrated that gH625 ameliorates the efficiency of liposomes to deliver PACAP to the mouse brain after intravenous administration. gH625-liposomes improve both PACAP reaching and crossing the BBB, as showed by the higher number of brain cells labelled with PACAP. gH625-liposomes represent a promising strategy to deliver therapeutic agents to CNS and to provide an effective imaging and diagnostic tool for the brain.

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.

Background: Stem cell-based therapies are an attractive option to promote regeneration and repair defective tissues and organs. Thanks to their multipotency, high proliferation rate and the lack of major ethical limitations, "olfactory ecto-mesenchymal stem cells" (OE-MSCs) have been described as a promising candidate to treat a variety of damaged tissues. Easily accessible in the nasal cavity of most mammals, these cells are highly suitable for autologous cell-based therapies and do not face issues associated with other stem cells. However, their clinical use in humans and animals is limited due to a lack of preclinical studies on autologous transplantation and because no well-established methods currently exist to cultivate these cells. Here we evaluated the feasibility of collecting, purifying and amplifying OE-MSCs from different mammalian genera with the goal of promoting their interest in veterinary regenerative medicine. Biopsies of olfactory mucosa from eight mammalian genera (mouse, rat, rabbit, sheep, dog, horse, gray mouse lemur and macaque) were collected, using techniques derived from those previously used in humans and rats. The possibility of amplifying these cells and their stemness features and differentiation capability were then evaluated., Results: Biopsies were successfully performed on olfactory mucosa without requiring the sacrifice of the donor animal, except mice. Cell populations were rapidly generated from olfactory mucosa explants. These cells displayed similar key features of their human counterparts: a fibroblastic morphology, a robust expression of nestin, an ability to form spheres and similar expression of surface markers (CD44, CD73). Moreover, most of them also exhibited high proliferation rates and clonogenicity with genus-specific properties. Finally, OE-MSCs also showed the ability to differentiate into mesodermal lineages., Conclusions: This article describes for the first time how millions of OE-MSCs can be quickly and easily obtained from different mammalian genera through protocols that are well-suited for autologous transplantations. Moreover, their multipotency makes them relevant to evaluate therapeutic application in a wide variety of tissue injury models. This study paves the way for the development of new fundamental and clinical studies based on OE-MSCs transplantation and suggests their interest in veterinary medicine.

After publication of the article [1], it has been brought to our attention that there are some errors in the formatting of names in the final version of the article.

LRP-1 is a large endocytic receptor mediating the clearance of various molecules from the extracellular matrix. LRP-1 was reported to control focal adhesion turnover to optimize the adhesion-deadhesion balance to support invasion. To better understand how LRP-1 coordinates cell-extracellular matrix interface, we explored its ability to regulate cell surface integrins in thyroid carcinomas. Using an antibody approach, we demonstrated that β1-integrin levels were increased at the plasma membrane under LRP1 silencing or upon RAP treatment, used as LRP-1 antagonist. Our data revealed that LRP-1 binds with both inactive and active β1-integrin conformations and identified the extracellular ligand-binding domains II or IV of LRP-1 as sufficient to bind β1-integrin. Using a recombinant β1-integrin, we demonstrated that LRP-1 acts as a regulator of β1-integrin intracellular traffic. Moreover, RAP or LRP-1 blocking antibodies decreased up to 36% the number of β1-integrin-containing endosomes. LRP-1 blockade did not significantly affect the levels of β1-integrin-containing lysosomes while decreasing localization of β1-integrin within Rab-11 positive vesicles. Overall, we identified an original molecular process in which LRP-1 acts as a main regulator of β1-integrin internalization and recycling in thyroid cancer cells., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

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., (© FASEB.)

Stem cell-based therapies critically rely on selective cell migration toward pathological or injured areas. We previously demonstrated that human olfactory ectomesenchymal stem cells (OE-MSCs), derived from an adult olfactory lamina propria, migrate specifically toward an injured mouse hippocampus after transplantation in the cerebrospinal fluid and promote functional recoveries. However, the mechanisms controlling their recruitment and homing remain elusive. Using an in vitro model of blood-brain barrier (BBB) and secretome analysis, we observed that OE-MSCs produce numerous proteins allowing them to cross the endothelial wall. Then, pan-genomic DNA microarrays identified signaling molecules that lesioned mouse hippocampus overexpressed. Among the most upregulated cytokines, both recombinant SPP1/osteopontin and CCL2/MCP-1 stimulate OE-MSC migration whereas only CCL2 exerts a chemotactic effect. Additionally, OE-MSCs express SPP1 receptors but not the CCL2 cognate receptor, suggesting a CCR2-independent pathway through other CCR receptors. These results confirm that OE-MSCs can be attracted by chemotactic cytokines overexpressed in inflamed areas and demonstrate that CCL2 is an important factor that could promote OE-MSC engraftment, suggesting improvement for future clinical trials.

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] c DPR) 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 K D 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 K D 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 10 6 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 K D 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 3 H-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.

Background: The heterogeneity of endothelial cell types underlies their remarkable ability to sub-specialize and provide specific requirements for a given vascular bed. Here, we compared rat microvascular endothelial cells (MECs) derived from the brain and spinal cord in both basal and inflammatory conditions., Methods: We used whole rat genome microarrays to compare, at different time points, basal and TNF-α-induced gene expression of rat MECs from in vitro models of the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB). Validation at both messenger RNA (mRNA) and protein levels was performed on freshly extracted microvessels (MVs) from the brain and spinal cord (BMVs and SCMVs, respectively), as these were considered the closest in vivo tissues to cultured MECs., Results: Most of the genes encoding adhesion/tight junction molecules and known endothelial markers were similarly expressed in brain and spinal cord MECs (BMECs and SCMECs, respectively). However, one striking finding was the higher expression of several Hox genes, which encode transcription factors involved in positional identity. The differential expression of Hoxa9 and Hoxb7 at the mRNA levels as well as protein levels was confirmed in BMVs and SCMVs. Although the TNF-α response was in general higher in BMECs than in SCMECs at 12 h, the opposite was observed at 48 h. Furthermore, we found that expression of Tnfrsf1a and Tnfrsf1b encoding the TNF receptor super-family member 1a/TNFR1 and 1b/TNFR2, respectively, were constitutively higher in BMVs compared to SCMVs. However, only Tnfrsf1b was induced in SCMECs in response to TNF-α at 24 and 48 h., Conclusions: Our results support a role for HOX members in defining the positional identities of MECs in vivo. Our data also suggest that the delayed transcriptional activation upon TNF-α treatment in SCMECs results from the requirement of the TNF-induced expression of Tnfrsf1b. In contrast, its high basal expression in BMECs might be sufficient to confer an immediate and efficient TNF-α response.

Pantethine, a natural low-molecular-weight thiol, shows a broad activity in a large range of essential cellular pathways. It has been long known as a hypolipidemic and hypocholesterolemic agent. We have recently shown that it exerts a neuroprotective action in mouse models of cerebral malaria and Parkinson's disease through multiple mechanisms. In the present study, we looked at its effects on membrane lipid rafts that serve as platforms for molecules engaged in cell activity, therefore providing a target against inappropriate cell response leading to a chronic inflammation. We found that pantethine-treated cells showed a significant change in raft fatty acid composition and cholesterol content, with ultimate downregulation of cell adhesion, CXCL12-driven chemotaxis, and transendothelial migration of various T cell types, including human Jurkat cell line and circulating effector T cells. The mechanisms involved include the alteration of the following: (i) CXCL12 binding to its target cells; (ii) membrane dynamics of CXCR4 and CXCR7, the two CXCL12 receptors; and (iii) cell redox status, a crucial determinant in the regulation of the chemokine system. In addition, we considered the linker for activation of T cells molecule to show that pantethine effects were associated with the displacement from the rafts of the acylated signaling molecules which had their palmitoylation level reduced.. In conclusion, the results presented here, together with previously published findings, indicate that due to its pleiotropic action, pantethine can downregulate the multifaceted process leading to pathogenic T cell activation and migration., (© 2015 Wiley Periodicals, Inc.)

The gene p8 encodes for a small cytoprotective protein with no apparent enzymatic activity being proposed to act as co-transcription factor whose expression is increased during inflammation. Recent data from astrocytes demonstrates that p8 suppression leads to induction of heme oxygenase 1 (HO-1). Here, we assessed the cross-talk between p8 and HO-1 in mouse embryonic fibroblasts (MEF) observing an increased expression of HO-1 in p8-deficient (p8(-/-)) MEFs in non-treated and treated conditions. This effect was independent of the cell cycle. Our findings revealed that generation of reactive oxygen species (ROS) was higher in p8(-/-) MEFs. Mitochondria and NADPH oxidases were not the origin of ROS. This observation was not restricted to MEF as suppression of p8 gene transcription in MiaPaCa-2 cells also led to increased intracellular ROS. Additionally, p8 deficiency did not affect the Rac1 dependant NADPH oxidase complex. Our data shows that p8 deficiency increases ROS and subsequently the expression of anti-oxidative enzymes, such as HO-1, suggesting an involvement in the anti-oxidative defense. Moreover, we suggest that the severity of AP observed in p8(-/-) mice is induced by an impaired anti oxidative capacity of the pancreas, which is caused by increased generation of ROS., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the extracellular matrix turnover by inhibiting the proteolytic activity of matrix metalloproteinases (MMPs). TIMP-1 also displays MMP-independent activities that influence the behavior of various cell types including neuronal plasticity, but the underlying molecular mechanisms remain mostly unknown. The trans-membrane receptor low-density lipoprotein receptor-related protein-1 (LRP-1) consists of a large extracellular chain with distinct ligand-binding domains that interact with numerous ligands including TIMP-2 and TIMP-3 and a short transmembrane chain with intracellular motifs that allow endocytosis and confer signaling properties to LRP-1. We addressed TIMP-1 interaction with recombinant ligand-binding domains of LRP-1 expressed by CHO cells for endocytosis study, or linked onto sensor chips for surface plasmon resonance analysis. Primary cortical neurons bound and internalized endogenous TIMP-1 through a mechanism mediated by LRP-1. This resulted in inhibition of neurite outgrowth and increased growth cone volume. Using a mutated inactive TIMP-1 variant we showed that TIMP-1 effect on neurone morphology was independent of its MMP inhibitory activity. We conclude that TIMP-1 is a new ligand of LRP-1 and we highlight a new example of its MMP-independent, cytokine-like functions.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm x cm(2) on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10(-3) cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Aim: The aim of this study was to develop poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) for delivery of a protein - tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) - across the blood-brain barrier (BBB) to inhibit deleterious matrix metalloproteinases (MMPs)., Materials and Methods: The NPs were formulated by multiple-emulsion solvent-evaporation, and for enhancing BBB penetration, they were coated with polysorbate 80 (Ps80). We compared Ps80-coated and uncoated NPs for their toxicity, binding, and BBB penetration on primary rat brain capillary endothelial cell cultures and the rat brain endothelial 4 cell line. These studies were followed by in vivo studies for brain delivery of these NPs., Results: Results showed that neither Ps80-coated nor uncoated NPs caused significant opening of the BBB, and essentially they were nontoxic. NPs without Ps80 coating had more binding to endothelial cells compared to Ps80-coated NPs. Penetration studies showed that TIMP-1 NPs + Ps80 had 11.21%± 1.35% penetration, whereas TIMP-1 alone and TIMP-1 NPs without Ps80 coating did not cross the endothelial monolayer. In vivo studies indicated BBB penetration of intravenously injected TIMP-1 NPs + Ps80., Conclusion: The study demonstrated that Ps80 coating of NPs does not cause significant toxic effects to endothelial cells and that it can be used to enhance the delivery of protein across endothelial cell barriers, both in vitro and in vivo.

The central nervous system (CNS) is protected by various barriers, which regulate nervous tissue homeostasis and control the selective and specific uptake, efflux, and metabolism of endogenous and exogenous molecules. Among these barriers is the blood-brain barrier (BBB), a physical and physiological barrier that filters very efficiently and selectively the entry of compounds from the blood to the brain and protects nervous tissue from harmful substances and infectious agents present in the bloodstream. The BBB also prevents the entry of potential drugs. As a result, various drug targeting and delivery strategies are currently being developed to enhance the transport of drugs from the blood to the brain. Following a general introduction, we briefly overview in this review article the fundamental physiological properties of the BBB. Then, we describe current strategies to bypass the BBB (i.e., invasive methods, alternative approaches, and temporary opening) and to cross it (i.e., noninvasive approaches). This section is followed by a chapter addressing the chemical and technological solutions developed to cross the BBB. A special emphasis is given to prodrug-targeting approaches and targeted nanotechnology-based systems, two promising strategies for BBB targeting and delivery of drugs to the brain., (© 2012 Wiley Periodicals, Inc.)

The decreasing number of approved drugs produced by the pharmaceutical industry, which has been accompanied by increasing expenses for R&D, demands alternative approaches to increase pharmaceutical R&D productivity. This situation has contributed to a revival of interest in peptides as potential drug candidates. New synthetic strategies for limiting metabolism and alternative routes of administration have emerged in recent years and resulted in a large number of peptide-based drugs that are now being marketed. This review reports on the unexpected and considerable number of peptides that are currently available as drugs and the chemical strategies that were used to bring them into the market. As demonstrated here, peptide-based drug discovery could be a serious option for addressing new therapeutic challenges., (Copyright 2009 Elsevier Ltd. All rights reserved.)