National Grants
Project's title: "Endothelial-targeted nanotherapy designed to silence receptor for advanced glycation end products (RAGE) and reduce inflammation in atherosclerosis"
Acronym: NANO-RAGE
Project's code: PN-II-RU-TE-2014-4-1837
Contract nr. 35/01.10.2015
Contract authority: Executive Agency or Higher Education Research Development and Innovation Funding (UEFISCDI)
Budget: 520.000 lei
Period: 1.10.2015-30.09.2017
Project's manager: Dr. Viorel Simion
Project's team:
Dr. Manuela Calin, cercetator senior
Dr. Elena Butoi, cercetator senior
Dr. Daniela Stan, postdoctorand
Drd. Cristina Ana Constantinescu, doctorand
Drd. Monica Madalina Tucureanu (Pirvulescu), doctorand
Geanina Voicu, masterand
Abstract:
Background. The receptors for advanced glycation end products (RAGE) expressed on endothelial cells (EC) play an essential role in the initiation and perpetuation of vascular wall inflammation in atherosclerosis. We hypothesize that to decrease the inflammatory process, specifically designed nanoparticles (NPs) can be developed and used as vectors for silencing RAGE in "inflamed" endothelium. The objective of NANO-RAGE project is to develop endothelium-targeted lipid nanoparticles loaded with RAGE-siRNA (lipoplexes) in order to decrease the vascular wall inflammation in atherosclerosis. Methods. We envisage to develop engineered cationic lipid nanocarriers for the entrapment of RAGE-siRNA: liposomes and solid lipid nanoparticles (SLN) that will have attached on their surface ligands that recognize a particular molecule expressed mainly by activated EC. The specific binding and fate of endothelial-targeted RAGE-siRNA/NPs lipoplexes and the silencing efficiency of RAGE receptors will be tested in vitro and in vivo employing cultured activated EC and an animal model of atherosclerosis (ApoE-deficient mice). Our project will introduce a novel targeted therapy and will provide important knowledge for developing strategies for better management of vascular diseases. The outcome of this project is highly relevant for nanomedicine field and holds the promise of a new targeted nanotherapeutic strategy to reduce vascular inflammation in atherosclerosis.
Expected results:
RAGE receptors (receptor for advanced glycation end products) play a key role in initiating and perpetuating inflammation in atherosclerosis, by their involvement in the recruitment and activation of inflammatory cells and in the activation of various intracellular signalling pathways in endothelial cells, a phenomenon that leads to increased secretion of pro-inflammatory cytokines and cell adhesion molecules. Thus, we believe that our innovative approach to silence RAGE expression will open new therapeutic perspectives for reducing vascular inflammation in atherosclerosis. The novelty of this study is the use of an adhesion molecule, P-selectin as an endothelial target for nanoparticles loaded with RAGE siRNA in order to manipulate the expression of RAGE in activated endothelial cells. Thus, we expect that the objectives of the project will provide innovative solutions based on nanotechnology to reduce the inflammation of vascular wall in atherosclerosis.
Phase I (Summary of results obtained)
Period: 1.10.2015-30.12.2015
Objective 1.1. Design and characterization of lipid nanoparticles carrying siRNA (siRNA/NP) targeted to activated vascular endothelium
Task 1.1.1: Preparation of siRNA/NP targeted to specific molecules expressed by activated endothelial cells (EC)
Task 1.1.2: Physico-chemical characterization of siRNA/NP
Receptors for advanced glycation end products (RAGE) expressed by endothelial cells (EC) plays an essential role in the initiation and perpetuation of vascular inflammation in atherosclerosis wall. Our hypothesis is that the inflammatory process can be inhibited using nanoparticles specially designed and used as vectors for silencing RAGE in the "inflamed" endothelium.
In this phase, cationic liposomes with various compositions of lipids were designed and characterized for size (by dynamic light scattering using NICOMP nanosizer) and for coupling efficiency to the surface of liposomes of a peptide with higher and specific affinity for the cell adhesion molecule P-selectin expressed on activated endothelial cells (by UHPLC).
Phase II (Summary of results obtained)
Period: 1.01.2016-15.12.2016
Objective 2.1. Evaluation of binding and cellular internalization of P-selectin targeted siRNA/NPs lipoplexes by activated endothelial cells
Task 2.1.1: Design and preparation of nanoparticles-encapsulated RAGE-siRNA (RAGE-siRNA/NPs) targeted to specific molecules expressed by activated endothelial cells (EC)
Task 2.1.2: Physico-chemical characterization of the RAGE-siRNA/NPs lipoplexes
Task 2.1.3: Screening for potent RAGE-siRNA by following the transfection efficiency of cultured EC
Task 2.1.4: Assessment of RAGE-siRNA/NPs lipoplexes cytotoxicity on EC
Task 2.1.5: Investigation of P-selectin targeted siRNA/NPs lipoplexes binding to activated EC
Task 2.1.6: Evaluation of the cellular internalization of P-selectin targeted siRNA/NPs lipoplexes by the activated EC in static and dynamic (simulating blood flow) conditions
The objective of phase 2 was to evaluate the targeting and internalization of lipid nanoparticles as carriers of siRNA (siRNA/NPs) designed to specifically recognize cell adhesion molecule P-selectin expressed on the surface of activated endothelial cells. Thus, targeted cationic liposomes directed to P-selectin by coupling peptides with high affinity for P-selectin or non-targeted cationic liposomes, coupled with peptides with random amino acid sequence (scrambled peptide) were designed and prepared. The binding and internalization of liposomes carrying siRNA directed to P-selectin (Psel-targeted siRNA/ NPs) were followed by fluorescence microscopy and flow cytometry using fluorescently labeled liposomes or siRNA, in static conditions and in conditions simulating the blood flow. There have also been investigated different siRNA sequences designed to silence the expression of RAGE receptor. The results are: 1) optimal composition of siRNA/ NPs that determine maximum efficiency of transfection and does not have significant cytotoxic effect was established; 2) lists of siRNA sequences designed for silencing human or murine RAGE receptor expression were built and the ability of siRNA sequences to silent RAGE expression was tested in mouse endothelial cells; 3) selective binding of siRNA/NPs targeted to P-selectin at b.End3 murine endothelial cells surface under dynamic incubation conditions, which simulates blood flow and 4) P-selectin targeted siRNA/NPs efficiently delivered siRNA into endothelial cells as compared to the commercial vectors. Also, internalisation of P-selectin targeted siRNA/NPs is significantly increased compared to internalisation of non-targeted siRNA/NPs, suggesting a specific uptake.
Dissemination
Book chapter:
1. Calin M, Butoi E, Manea SA, Simionescu M, Manea A (2016). Lessons from experimental-induced atherosclerosis – valuable for the precision medicine of tomorrow. In book: Arterial Revascularization of the Head and Neck: Text Atlas for Prevention and Management of Stroke, pp.341-365, Editor Horia Muresian, Springer, New York, DOI: 10.1007/978-3-319-34193-4_17, ISBN-10: 331934191X, ISBN-13: 978-3319341910.
ISI papers:
1. Simion V, Constantinescu CA, Stan D, Deleanu M, Tucureanu MM, Butoi E, Manduteanu I, Simionescu M, Calin M, P-Selectin Targeted Dexamethasone-Loaded Lipid Nanoemulsions: A Novel Therapy to Reduce Vascular Inflammation, Mediators of Inflammation. 2016;2016:1625149 (I.F. 3.418).
2. Calin M, Manduteanu I, Emerging Nanocarriers-Based Approaches To Diagnose And Reduce Vascular Inflammation In Atherosclerosis, Current Medicinal Chemistry, 2016 Nov 22, [Epub ahead of print], PMID: 27881067 (I.F. 3.455).
