title: Layer-by-layer nanoparticles as an efficient siRNA delivery vehicle for SPARC silencing.
abstract: Efficient and safe delivery systems for siRNA therapeutics remain a challenge. Elevated secreted protein, acidic, and rich in cysteine (SPARC) protein expression is associated with tissue scarring and fibrosis. Here we investigate the feasibility of encapsulating SPARC-siRNA in the bilayers of layer-by-layer (LbL) nanoparticles (NPs) with poly(L-arginine) (ARG) and dextran (DXS) as polyelectrolytes. Cellular binding and uptake of LbL NPs as well as siRNA delivery were studied in FibroGRO cells. siGLO-siRNA and SPARC-siRNA were efficiently coated onto hydroxyapatite nanoparticles. The multilayered NPs were characterized with regard to particle size, zeta potential and surface morphology using dynamic light scattering and transmission electron microscopy. The SPARC-gene silencing and mRNA levels were analyzed using ChemiDOC western blot technique and RT-PCR. The multilayer SPARC-siRNA incorporated nanoparticles are about 200 nm in diameter and are efficiently internalized into FibroGRO cells. Their intracellular fate was also followed by tagging with suitable reporter siRNA as well as with lysotracker dye; confocal microscopy clearly indicates endosomal escape of the particles. Significant (60%) SPARC-gene knock down was achieved by using 0.4 pmole siRNA/μg of LbL NPs in FibroGRO cells and the relative expression of SPARC mRNA reduced significantly (60%) against untreated cells. The cytotoxicity as evaluated by xCelligence real-time cell proliferation and MTT cell assay, indicated that the SPARC-siRNA-loaded LbL NPs are non-toxic. In conclusion, the LbL NP system described provides a promising, safe and efficient delivery platform as a non-viral vector for siRNA delivery that uses biopolymers to enhance the gene knock down efficiency for the development of siRNA therapeutics.
authors: Singapore Eye Research Institute, 11 Third Hospital Avenue, 168751, Singapore.
authors: Yang Fei
authors: YF
authors: Tan
authors: Raghavendra C
authors: RC
authors: Mundargi
authors: Min Hui Averil
authors: MH
authors: Chen
authors: Jacqueline
authors: J
authors: Lessig
authors: Björn
authors: B
authors: Neu
authors: Subbu S
authors: SS
authors: Venkatraman
authors: Tina T
authors: TT
authors: Wong
mesh_terms: D004705:Endocytosis; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008297:Male; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D009363:Neoplasm Proteins / Q000378:metabolism; D015676:Osteonectin / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000648:ultrastructure; D010316:Particle Size; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism; D055672:Static Electricity
chemical_list: D009363:Neoplasm Proteins; D015676:Osteonectin; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C085488:RPL13 protein, human; D012269:Ribosomal Proteins
keywords: FibroGRO; Layer-by-layer nanoparticles; SPARC-siRNA; glaucoma; siGLO