Long Noncoding RNAs as Therapeutic Targets.

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of cellular functions including maintenance of cellular homeostasis as well as the onset and progression of disease. LncRNAs often exhibit cell-, tissue-, and disease-specific expression patterns, making them desirable therapeutic targets. LncRNAs are commonly targeted using oligonucleotide therapeutics, and advances in oligonucleotide chemistry including C2 ribose sugar modifications such as 2'-fluoro, 2'-O-methyl, and 2-O-methoxyethyl modifications; 2'4'-constrained nucleotides such as locked nucleic acids and constrained 2'-O-ethyl (cEt) nucleotides; and phosphorothioate bonds have dramatically improved efficacy of oligonucleotide therapies. Novel delivery platforms such as viral vectors and nanoparticles have also improved pharmacokinetic properties of oligonucleotides targeting lncRNAs. Accumulating pre-clinical studies have utilized these strategies to therapeutically target lncRNAs and alter progression of many different disease states including Snhg12 and Chast in cardiovascular disease, Mirt2 and HOTTIP in sepsis and autoimmune disease, and Malat1 and HOXB-AS3 in cancer. Emerging oligonucleotide conjugation methods including the use of peptide nucleic acids hold promise to facilitate targeting to specific tissue types. Here, we review recent advances in lncRNA therapeutics and provide examples of how lncRNAs have been successfully targeted in pre-clinical models of disease. Finally, we detail remaining challenges facing the lncRNA field and how advances in delivery platforms and oligonucleotide chemistry might help overcome these barriers to catalyze the translation of pre-clinical studies to successful pharmaceutical development.

LentiRILES, a miRNA-ON sensor system for monitoring the functionality of miRNA in cancer biology and therapy.

A major unresolved challenge in miRNA biology is the capacity to monitor the spatiotemporal activity of miRNAs expressed in animal disease models. We recently reported that the miRNA-ON monitoring system called RILES (RNAi-inducible expression Luciferase system) implanted in lentivirus expression system (LentiRILES) offers unique opportunity to decipher the kinetics of miRNA activity in vitro, in relation with their intracellular trafficking in glioblastoma cells. In this study, we describe in detail the method for the production of LentiRILES stable cell lines and employed it in several applications in the field of miRNA biology and therapy. We show that LentiRILES is a robust, highly specific and sensitive miRNA sensor system that can be used in vitro as a single-cell miRNA monitoring method, cell-based screening platform for miRNA therapeutics and as a tool to analyse the structure-function relationship of the miRNA duplex. Furthermore, we report the kinetics of miRNA activity upon the intracranial delivery of miRNA mimics in an orthotopic animal model of glioblastoma. This information is exploited to evaluate the tumour suppressive function of miRNA-200c as locoregional therapeutic modality to treat glioblastoma. Our data provide evidence that LentiRILES is a robust system, well suited to resolve the activity of endogenous and exogenously expressed miRNAs from basic research to gene and cell therapy.

A Smooth Muscle Cell-Enriched Long Noncoding RNA Regulates Cell Plasticity and Atherosclerosis by Interacting With Serum Response Factor.

Objective: Vascular smooth muscle cell (VSMC) plasticity plays a critical role in the development of atherosclerosis. Long noncoding RNAs (lncRNAs) are emerging as important regulators in the vessel wall and impact cellular function through diverse interactors. However, the role of lncRNAs in regulating VSMCs plasticity and atherosclerosis remains unclear. Approach and Results: We identified a VSMC-enriched lncRNA cardiac mesoderm enhancer-associated noncoding RNA (CARMN) that is dynamically regulated with progression of atherosclerosis. In both mouse and human atherosclerotic plaques, CARMN colocalized with VSMCs and was expressed in the nucleus. Knockdown of CARMN using antisense oligonucleotides in Ldlr−/− mice significantly reduced atherosclerotic lesion formation by 38% and suppressed VSMCs proliferation by 45% without affecting apoptosis. In vitro CARMN gain- and loss-of-function studies verified effects on VSMC proliferation, migration, and differentiation. TGF-ß1 (transforming growth factor-beta) induced CARMN expression in a Smad2/3-dependent manner. CARMN regulated VSMC plasticity independent of the miR143/145 cluster, which is located in close proximity to the CARMN locus. Mechanistically, lncRNA pulldown in combination with mass spectrometry analysis showed that the nuclear-localized CARMN interacted with SRF (serum response factor) through a specific 600­1197 nucleotide domain. CARMN enhanced SRF occupancy on the promoter regions of its downstream VSMC targets. Finally, knockdown of SRF abolished the regulatory role of CARMN in VSMC plasticity. Conclusions: The lncRNA CARMN is a critical regulator of VSMC plasticity and atherosclerosis. These findings highlight the role of a lncRNA in SRF-dependent signaling and provide implications for a range of chronic vascular occlusive disease states.

LncRNA-MAP3K4 regulates vascular inflammation through the p38 MAPK signaling pathway and cis-modulation of MAP3K4.

Chronic vascular inflammation plays a key role in the pathogenesis of atherosclerosis. Long non-coding RNAs (lncRNAs) have emerged as essential inflammation regulators. We identify a novel lncRNA termed lncRNA-MAP3K4 that is enriched in the vessel wall and regulates vascular inflammation. In the aortic intima, lncRNA-MAP3K4 expression was reduced by 50% during the progression of atherosclerosis (chronic inflammation) and 70% during endotoxemia (acute inflammation). lncRNA-MAP3K4 knockdown reduced the expression of key inflammatory factors (eg, ICAM-1, E-selectin, MCP-1) in endothelial cells or vascular smooth muscle cells and decreased monocytes adhesion to endothelium, as well as reducing TNF-α, IL-1ß, COX2 expression in macrophages. Mechanistically, lncRNA-MAP3K4 regulates inflammation through the p38 MAPK signaling pathway. lncRNA-MAP3K4 shares a bidirectional promoter with MAP3K4, an upstream regulator of the MAPK signaling pathway, and regulates its transcription in cis. lncRNA-MAP3K4 and MAP3K4 show coordinated expression in response to inflammation in vivo and in vitro. Similar to lncRNA-MAP3K4, MAP3K4 knockdown reduced the expression of inflammatory factors in several different vascular cells. Furthermore, lncRNA-MAP3K4 and MAP3K4 knockdown showed cooperativity in reducing inflammation in endothelial cells. Collectively, these findings unveil the role of a novel lncRNA in vascular inflammation by cis-regulating MAP3K4 via a p38 MAPK pathway.

A macrophage-specific lncRNA regulates apoptosis and atherosclerosis by tethering HuR in the nucleus.

Long non-coding RNAs (lncRNAs) are emerging regulators of pathophysiological processes including atherosclerosis. Using RNA-seq profiling of the intima of lesions, here we identify a macrophage-specific lncRNA MAARS (Macrophage-Associated Atherosclerosis lncRNA Sequence). Aortic intima expression of MAARS increases by 270-fold with atherosclerotic progression and decreases with regression by 60%. MAARS knockdown reduces atherosclerotic lesion formation by 52% in LDLR-/- mice, largely independent of effects on lipid profile and inflammation, but rather by decreasing macrophage apoptosis and increasing efferocytosis in the vessel wall. MAARS interacts with HuR/ELAVL1, an RNA-binding protein and important regulator of apoptosis. Overexpression and knockdown studies verified MAARS as a critical regulator of macrophage apoptosis and efferocytosis in vitro, in an HuR-dependent manner. Mechanistically, MAARS knockdown alters HuR cytosolic shuttling, regulating HuR targets such as p53, p27, Caspase-9, and BCL2. These findings establish a mechanism by which a macrophage-specific lncRNA interacting with HuR regulates apoptosis, with implications for a broad range of vascular disease states.

Computational Analysis of Targeting SARS-CoV-2, Viral Entry Proteins ACE2 and TMPRSS2, and Interferon Genes by Host MicroRNAs.

Rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has led to a global pandemic, failures of local health care systems, and global economic recession. MicroRNAs (miRNAs) have recently emerged as important regulators of viral pathogenesis, particularly among RNA viruses, but the impact of host miRNAs on SARS-CoV-2 infectivity remains unknown. In this study, we utilize the combination of powerful bioinformatic prediction algorithms and miRNA profiling to predict endogenous host miRNAs that may play important roles in regulating SARS-CoV-2 infectivity. We provide a collection of high-probability miRNA binding sites within the SARS-CoV-2 genome as well as within mRNA transcripts of critical viral entry proteins ACE2 and TMPRSS2 and their upstream modulators, the interferons (IFN). By utilizing miRNA profiling datasets of SARS-CoV-2-resistant and -susceptible cell lines, we verify the biological plausibility of the predicted miRNA-target RNA interactions. Finally, we utilize miRNA profiling of SARS-CoV-2-infected cells to identify predicted miRNAs that are differentially regulated in infected cells. In particular, we identify predicted miRNA binders to SARS-CoV-2 ORFs (miR-23a (1ab), miR-29a, -29c (1ab, N), miR-151a, -151b (S), miR-4707-3p (S), miR-298 (5'-UTR), miR-7851-3p (5'-UTR), miR-8075 (5'-UTR)), ACE2 3'-UTR (miR-9-5p, miR-218-5p), TMPRSS2 3'-UTR (let-7d-5p, -7e-5p, miR-494-3p, miR-382-3p, miR-181c-5p), and IFN-α 3'-UTR (miR-361-5p, miR-410-3p). Overall, this study provides insight into potential novel regulatory mechanisms of SARS-CoV-2 by host miRNAs and lays the foundation for future investigation of these miRNAs as potential therapeutic targets or biomarkers.

LncRNA VINAS regulates atherosclerosis by modulating NF-κB and MAPK signaling.

Long noncoding RNAs (lncRNAs) play important roles in regulating diverse cellular processes in the vessel wall, including atherosclerosis. RNA-Seq profiling of intimal lesions revealed a lncRNA, VINAS (Vascular INflammation and Atherosclerosis lncRNA Sequence), that is enriched in the aortic intima and regulates vascular inflammation. Aortic intimal expression of VINAS fell with atherosclerotic progression and rose with regression. VINAS knockdown reduced atherosclerotic lesion formation by 55% in LDL receptor-deficient (LDLR-/-) mice, independent of effects on circulating lipids, by decreasing inflammation in the vessel wall. Loss- and gain-of-function studies in vitro demonstrated that VINAS serves as a critical regulator of inflammation by modulating NF-κB and MAPK signaling pathways. VINAS knockdown decreased the expression of key inflammatory markers, such as MCP-1, TNF-α, IL-1ß, and COX-2, in endothelial cells (ECs), vascular smooth muscle cells, and bone marrow-derived macrophages. Moreover, VINAS silencing decreased expression of leukocyte adhesion molecules VCAM-1, E-selectin, and ICAM-1 and reduced monocyte adhesion to ECs. DEP domain containing 4 (DEPDC4), an evolutionary conserved human ortholog of VINAS with approximately 74% homology, showed similar regulation in human and pig atherosclerotic specimens. DEPDC4 knockdown replicated antiinflammatory effects of VINAS in human ECs. These findings reveal a potentially novel lncRNA that regulates vascular inflammation, with broad implications for vascular diseases.

Intracellular trafficking and functional monitoring of miRNA delivery in glioblastoma using lipopolyplexes and the miRNA-ON RILES reporter system.

MicroRNA (miRNA) oligonucleotides therapeutics are potent and attractive drugs for cancer treatment, but the kinetics of their intracellular trafficking, RISC processing and interaction with their mRNA targets in the cells are still not well understood. Moreover, the absence of efficient carriers impairs their translation into the clinic. Here, we compare the kinetics of miRNA-133a activity after transfection of U87MG glioblastoma cells with either a home-made lipopolyplexes (LPRi) or with the RNAiMax transfection reagent. For this purpose, we combined miRNA intracellular trafficking studies by confocal microscopy with our previously described RILES miRNA-ON reporter system subcloned here in a lentivirus expression vector (LentiRILES) for longitudinal analysis of miRNA activity in transfected cells. Using the LentiRILES system, we report significant differences in terms of miRNA delivery kinetics performed by these two transfection regents. We decipher the mechanisms of miRNA delivery by LPRi and investigate the main steps of miRNA internalization and cytosolic processing. We demonstrate that LPRi preferentially uses caveolae-mediated endocytosis as the main internalization pathway, releases miRNA into the cytosol after the first 3 h of incubation, and addresses the cytosolic miRNAs to P-bodies, while a fraction of miRNAs are exported to the extracellular space through exosomes which were found fully capable to re-transfect the cells. We implanted the LentiRILES cells in the brain of mice and infused the tumours with LPRi.miRNA using the convection-enhanced delivery method. Bioluminescence imaging of the live mice revealed efficient delivery of miRNAs in glioblastoma tumours, attesting successful miRNA uptake, internalization and RISC activation in vivo. Overall, our study provides a comprehensive overview of miRNA intracellular trafficking and processing in a glioblastoma context and highlights the potential use of LPRi for miRNA-based therapy.

Long noncoding RNA SNHG12 integrates a DNA-PK-mediated DNA damage response and vascular senescence.

Long noncoding RNAs (lncRNAs) are emerging regulators of biological processes in the vessel wall; however, their role in atherosclerosis remains poorly defined. We used RNA sequencing to profile lncRNAs derived specifically from the aortic intima of Ldlr -/- mice on a high-cholesterol diet during lesion progression and regression phases. We found that the evolutionarily conserved lncRNA small nucleolar host gene-12 (SNHG12) is highly expressed in the vascular endothelium and decreases during lesion progression. SNHG12 knockdown accelerated atherosclerotic lesion formation by 2.4-fold in Ldlr -/- mice by increased DNA damage and senescence in the vascular endothelium, independent of effects on lipid profile or vessel wall inflammation. Conversely, intravenous delivery of SNHG12 protected the tunica intima from DNA damage and atherosclerosis. LncRNA pulldown in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that SNHG12 interacted with DNA-dependent protein kinase (DNA-PK), an important regulator of the DNA damage response. The absence of SNHG12 reduced the DNA-PK interaction with its binding partners Ku70 and Ku80, abrogating DNA damage repair. Moreover, the anti-DNA damage agent nicotinamide riboside (NR), a clinical-grade small-molecule activator of NAD+, fully rescued the increases in lesional DNA damage, senescence, and atherosclerosis mediated by SNHG12 knockdown. SNHG12 expression was also reduced in pig and human atherosclerotic specimens and correlated inversely with DNA damage and senescent markers. These findings reveal a role for this lncRNA in regulating DNA damage repair in the vessel wall and may have implications for chronic vascular disease states and aging.

Targeted Transfection Using PEGylated Cationic Liposomes Directed Towards P-Selectin Increases siRNA Delivery into Activated Endothelial Cells.

: The progress in small-interfering RNA (siRNA) therapeutics depends on the development of suitable nanocarriers to perform specific and effective delivery to dysfunctional cells. In this paper, we questioned whether P-selectin, a cell adhesion molecule specifically expressed on the surface of activated endothelial cells (EC) could be employed as a target for nanotherapeutic intervention. To this purpose, we developed and characterized P-selectin targeted PEGylated cationic liposomes able to efficiently pack siRNA and to function as efficient vectors for siRNA delivery to tumour necrosis factor-α (TNF-α) activated EC. Targeted cationic liposomes were obtained by coupling a peptide with high affinity for P-selectin to a functionalized PEGylated phospholipid inserted in the liposomes' bilayer (Psel-lipo). As control, scrambled peptide coupled cationic liposomes (Scr-lipo) were used. The lipoplexes obtained by complexation of Psel-lipo with siRNA (Psel-lipo/siRNA) were taken up specifically and at a higher extent by TNF-α activated b.End3 endothelial cells as compared to non-targeted Scr-lipo/siRNA. The Psel-lipo/siRNA delivered with high efficiency siRNA into the cells. The lipoplexes were functional as demonstrated by the down-regulation of the selected gene (GAPDH). The results demonstrate an effective targeted delivery of siRNA into cultured activated endothelial cells using P-selectin directed PEGylated cationic liposomes, which subsequently knock-down the desired gene.

LncRNAs in vascular biology and disease.

Accumulating studies indicate that long non-coding RNAs (lncRNAs) play important roles in the regulation of diverse biological processes involved in homeostatic control of the vessel wall in health and disease. However, our knowledge of the mechanisms by which lncRNAs control gene expression and cell signaling pathways is still nascent. Furthermore, only a handful of lncRNAs has been functionally evaluated in response to pathophysiological stimuli or in vascular disease states. For example, lncRNAs may regulate endothelial dysfunction by modulating endothelial cell proliferation (e.g. MALAT1, H19) or angiogenesis (e.g. MEG3, MANTIS). LncRNAs have also been implicated in modulating vascular smooth muscle cell (VSMC) phenotypes or vascular remodeling (e.g. ANRIL, SMILR, SENCR, MYOSLID). Finally, emerging studies have implicated lncRNAs in leukocytes activation (e.g. lincRNA-Cox2, linc00305, THRIL), macrophage polarization (e.g. GAS5), and cholesterol metabolism (e.g. LeXis). This review summarizes recent findings on the expression, mechanism, and function of lncRNAs implicated in a range of vascular disease states from mice to human subjects. An improved understanding of lncRNAs in vascular disease may provide new pathophysiological insights and opportunities for the generation of a new class of RNA-based biomarkers and therapeutic targets.

Long Non-Coding RNAs in Vascular Inflammation.

Less than 2% of the genome encodes for proteins. Accumulating studies have revealed a diverse set of RNAs derived from the non-coding genome. Among them, long non-coding RNAs (lncRNAs) have garnered widespread attention over recent years as emerging regulators of diverse biological processes including in cardiovascular disease (CVD). However, our knowledge of their mechanisms by which they control CVD-related gene expression and cell signaling pathways is still limited. Furthermore, only a handful of lncRNAs has been functionally evaluated in the context of vascular inflammation, an important process that underlies both acute and chronic disease states. Because some lncRNAs may be expressed in cell- and tissue-specific expression patterns, these non-coding RNAs hold great promise as novel biomarkers and as therapeutic targets in health and disease. Herein, we review those lncRNAs implicated in pro- and anti-inflammatory processes of acute and chronic vascular inflammation. An improved understanding of lncRNAs in vascular inflammation may provide new pathophysiological insights in CVD and opportunities for the generation of a new class of RNA-based biomarkers and therapeutic targets.

Long noncoding RNAs in cardiovascular disease, diagnosis, and therapy.

PURPOSE OF REVIEW: Long noncoding RNAs (lncRNAs) have emerged as powerful regulators of nearly all biological processes. Their cell-type and tissue-specific expression in health and disease provides new avenues for diagnosis and therapy. This review highlights the role of lncRNAs that are involved in cardiovascular disease (CVD) with a special focus on cell types involved in cardiac injury and remodeling, vascular injury, angiogenesis, inflammation, and lipid metabolism. RECENT FINDINGS: Almost 98% of the genome does not encode for proteins. LncRNAs are among the most abundant type of RNA in the noncoding genome. Accumulating studies have uncovered novel lncRNA-mediated regulation of CVD-associated genes, signaling pathways, and pathophysiological responses. Targeting lncRNAs in vivo using short antisense oligonucleotides or by gene editing has provided important insights into disease pathogenesis through epigenetic, transcriptional, or translational mechanisms. Although cross-species conservation still remains a major obstacle, there is increasing appreciation that altered expression of lncRNAs associates with stage-specific CVD and in human patient cohorts, providing new opportunities for diagnosis and therapy. SUMMARY: A better understanding of lncRNAs will not only fundamentally improve our understanding of key signaling pathways in CVD, but also aid in the development of effective new therapies and RNA-based biomarkers.

Positive radionuclide imaging of miRNA expression using RILES and the human sodium iodide symporter as reporter gene is feasible and supports a protective role of miRNA-23a in response to muscular atrophy.

MicroRNAs (miRNAs) are key players in many biological processes and are considered as an emerging class of pharmacology drugs for diagnosis and therapy. However to fully exploit the therapeutic potential of miRNAs, it is becoming crucial to monitor their expression pattern using medical imaging modalities. Recently, we developed a method called RILES, for RNAi-Inducible Luciferase Expression System that relies on an engineered regulatable expression system to switch-ON the expression of the luciferase gene when a miRNA of interest is expressed in cells. Here we investigated whether replacing the luciferase reporter gene with the human sodium iodide symporter (hNIS) reporter gene will be also suited to monitor the expression of miRNAs in a clinical setting context. We provide evidence that radionuclide imaging of miRNA expression using hNIS is feasible although it is not as robust as when the luciferase reporter gene is used. However, under appropriate conditions, we monitored the expression of several miRNAs in cells, in the liver and in the tibialis anterior muscle of mice undergoing muscular atrophy. We demonstrated that radiotracer accumulation in transfected cells correlated with the induction of hNIS and with the expression of miRNAs detected by real time PCR. We established the kinetic of miRNA-23a expression in mice and demonstrated that this miRNA follows a biphasic expression pattern characterized by a loss of expression at a late time point of muscular atrophy. At autopsy, we found an opposite expression pattern between miRNA-23a and one of the main transcriptional target of this miRNA, APAF-1, and as downstream target, Caspase 9. Our results report the first positive monitoring of endogenously expressed miRNAs in a nuclear medicine imaging context and support the development of additional work to establish the potential therapeutic value of miRNA-23 to prevent the damaging effects of muscular atrophy.

MicroRNAs in Neurocognitive Dysfunctions: New Molecular Targets for Pharmacological Treatments?

BACKGROUND: Neurodegenerative and cognitive disorders are multifactorial diseases (i.e., involving neurodevelopmental, genetic, age or environmental factors) characterized by an abnormal development that affects neuronal function and integrity. Recently, an increasing number of studies revealed that the dysregulation of microRNAs (miRNAs) may be involved in the etiology of cognitive disorders as Alzheimer, Parkinson, and Huntington's diseases, Schizophrenia and Autism spectrum disorders. METHODS: From an extensive search in bibliographic databases of peer-reviewed research literature, we identified relevant published studies related to specific key words such as memory, cognition, neurodegenerative disorders, neurogenesis and miRNA. We then analysed, evaluated and summerized scientific evidences derived from these studies. RESULTS: We first briefly summarize the basic molecular events involved in memory, a process inherent to cognitive disease, and then describe the role of miRNAs in neurodevelopment, synaptic plasticity and memory. Secondly, we provide an overview of the impact of miRNA dysregulation in the pathogenesis of different neurocognitive disorders, and lastly discuss the feasibility of miRNA-based therapeutics in the treatment of these disorders. CONCLUSION: This review highlights the molecular basis of neurodegenerative and cognitive disorders by focusing on the impact of miRNAs dysregulation in these pathological phenotypes. Altogether, the published reports suggest that miRNAs-based therapy could be a viable therapeutic alternative to current treatment options in the future.

Pharmacomodulation of microRNA Expression in Neurocognitive Diseases: Obstacles and Future Opportunities.

Given the importance of microRNAs (miRNAs) in modulating brain functions and their implications in neurocognitive disorders there are currently significant efforts devoted in the field of miRNA-based therapeutics to correct and/or to treat these brain diseases. The observation that miRNA 29a/b-1 cluster, miRNA 10b and miRNA 7, for instance, are frequently deregulated in the brains of patients with neurocognitive diseases and in animal models of Alzheimer, Huntington's and Parkinson's diseases, suggest that correction of miRNA expression using agonist or antagonist miRNA oligonucleotides might be a promising approach to correct or even to cure such diseases. The encouraging results from recent clinical trials allow envisioning that pharmacological approaches based on miRNAs might, in a near future, reach the requirements for successful therapeutic outcomes and will improve the healthcare of patients with brain injuries or disorders. This review will focus on the current strategies used to modulate pharmacological function of miRNA using chemically modified oligonucleotides. We will then review the recent literature on strategies to improve nucleic acid delivery across the blood-brain barrier which remains a severe obstacle to the widespread application of miRNA therapeutics to treat brain diseases. Finally, we provide a state-of-art of current preclinical research performed in animal models for the treatment of neurocognitive disorders using miRNA as therapeutic agents and discuss future developments of miRNA therapeutics.

P-Selectin Targeted Dexamethasone-Loaded Lipid Nanoemulsions: A Novel Therapy to Reduce Vascular Inflammation.

Inflammation is a common process associated with numerous vascular pathologies. We hypothesized that targeting the inflamed endothelium by coupling a peptide with high affinity for P-selectin to the surface of dexamethasone-loaded lipid nanoemulsions will highly increase their specific binding to activated endothelial cells (EC) and reduce the cell activation. We developed and characterized dexamethasone-loaded lipid nanoemulsions directed towards P-selectin (PLN-Dex) and monitored their anti-inflammatory effects in vitro using cultured EC (EA.hy926 cells) and in vivo using a mouse model of acute inflammation [lipopolysaccharides (LPS) intravenously administered in C57BL/6 mice]. We found that PLN-Dex bound specifically to the surface of activated EC are efficiently internalized by EC and reduced the expression of proinflammatory genes, thus preventing the monocyte adhesion and transmigration to/through activated EC. Given intravenously in mice with acute inflammation, PLN-Dex accumulated at a significant high level in the lungs (compared to nontargeted nanoemulsions) and significantly reduced mRNA expression level of key proinflammatory cytokines such as IL-1ß, IL-6, and MCP-1. In conclusion, the newly developed nanoformulation, PLN-Dex, is functional in vitro and in vivo, reducing selectively the endothelium activation and the consequent monocyte infiltration and diminishing significantly the lungs' inflammation, in a mouse model of acute inflammation.

Conjugation of curcumin-loaded lipid nanoemulsions with cell-penetrating peptides increases their cellular uptake and enhances the anti-inflammatory effects in endothelial cells.

OBJECTIVES: To prepare and characterize in vitro and in vivo lipid nanoemulsions (LN) loaded with curcumin (Cm) and functionalized with a cell-penetrating peptide. METHODS: Curcumin-loaded lipid nanoemulsions (CmLN) functionalized with a nona-arginine peptide (R9-CmLN) have been obtained, characterized and optimized for size, entrapment efficiency and in vitro Cm release. The interaction of R9-CmLN with human endothelial cells (HEC) was investigated using cultured EA.hy926 cells, and in vivo biodistribution studies were performed using C57BL6 mice. KEY FINDINGS: When used in therapeutically relevant concentration, R9-CmLN have low haemolytic activity, low cytotoxicity on HEC, and show anti-inflammatory effects by reducing the monocytes adhesion to TNF-α activated HEC. Moreover, HEC uptake and internalization of R9-CmLN was significantly higher compared to the non-functionalized CmLN. In vivo biodistribution studies in mice revealed a higher accumulation of R9-CmLN in the liver and the lungs compared to CmLN and the body clearance of the both nanoformulations after 72 h. CONCLUSIONS: Cell-penetrating peptides-functionalized CmLN have superior characteristics compared to their non-functionalized counterparts: are more efficiently internalized by the cells, produces anti-inflammatory effects in HEC and when administrated intravenously in mice exhibit increased accumulation in the liver and the lungs, suggesting their potential therapeutic applications in different inflammatory pathologies localized in the liver or the lungs.

Hybrid fullerene conjugates as vectors for DNA cell-delivery.

The present study reports fullerene conjugates that act as efficient binders of double stranded DNA (dsDNA) into cytofriendly polyplexes. The conjugates are designed to generate dendrimeric structures, having C60 as the core and bearing linear or branched PEI and polyethyleneglycol (PEG) arms (∼2 kDa). Simple and reproducible synthesis pathways provided C60-PEI and C60-PEG-PEI conjugates. They were able to bind linear and plasmidic dsDNA and they form particulate polyplexes of 50 to 200 nm in diameter. The resulted polyplexes toggle between the anionic and cationic state at nitrogen to phosphorous ratios (N/P) of about 5, as revealed by their zeta potential and became colloidally stable at N/P ratios above 10, as determined by atomic force microscopy (AFM). They are electrophoretically unbreakable starting with N/P ratios of 3 and of 5 when salmon sperm DNA and pEYFP-C1 plasmid, respectively are loaded. Both C60-PEI·pEYFP and C60-PEG-PEI·pEYFP polyplexes are non-cytotoxic against HEK 293T cells in culture and exhibit transfection efficiency better than 25% (N/P ratios above 20) and 6% (N/P ratios above 60) respectively, measured by flow cytometry. For comparison, the commercial SuperFect® from Qiagen (positive control) was able to provide an efficiency of 15-20%, under similar conditions. Moreover, the C60-PEG-PEI conjugate is as performant as the positive control in terms of expression of EYFP reporter gene in cultured cells and exhibited high cytocompatibility, determining cell proliferation up to 200%. Our study proved that C60-PEG-PEI is effective vector for DNA delivery being, in addition, easily synthesizable, practically non-cytotoxic and as efficient the commercially available transfection tools.

VCAM-1 directed target-sensitive liposomes carrying CCR2 antagonists bind to activated endothelium and reduce adhesion and transmigration of monocytes.

Chemokines are critically involved in the development of chronic inflammatory-associated diseases such as atherosclerosis. We hypothesized that targeted delivery of compounds to the surface of activated endothelial cells (EC) interferes with chemokine/receptor interaction and thereby efficiently blocks inflammation. We developed PEGylated target-sensitive liposomes (TSL) encapsulating a CCR2 antagonist (Teijin compound 1) coupled with a specific peptide recognized by endothelial VCAM-1 (Vp-TSL-Tj). TSL were characterized for size (by dynamic light scattering), the amount of peptide coupled at the liposomal surface and Teijin release (by HPLC). We report that Vp-TSL-Tj binds specifically to activated EC in vitro and in situ, release the entrapped Teijin and prevent the transmigration of monocytes through activated EC. This is the first evidence that nanocarriers which transport and release chemokine inhibitors at specific pathological sites can reduce chemokine-dependent inflammatory processes.