An in situ gelling liquid crystalline system based on monoglycerides and polyethylenimine for local delivery of siRNAs.

The development of delivery systems able to complex and release siRNA into the cytosol is essential for therapeutic use of siRNA. Among the delivery systems, local delivery has advantages over systemic administration. In this study, we developed and characterized non-viral carriers to deliver siRNA locally, based on polyethylenimine (PEI) as gene carrier, and a self-assembling drug delivery system that forms a gel in situ. Liquid crystalline formulations composed of monoglycerides (MO), PEI, propylene glycol (PG) and 0.1M Tris buffer pH 6.5 were developed and characterized by polarized light microscopy, Small Angle X-ray Scattering (SAXS), for their ability to form inverted type liquid crystalline phases (LC2) in contact with excess water, water absorption capacity, ability to complex with siRNA and siRNA release. In addition, gel formation in vivo was determined by subcutaneous injection of the formulations in mice. In water excess, precursor fluid formulations rapidly transformed into a viscous liquid crystalline phase. The presence of PEI influences the liquid crystalline structure of the LC2 formed and was crucial for complexing siRNA. The siRNA was released from the crystalline phase complexed with PEI. The release rate was dependent on the rate of water uptake. The formulation containing MO/PEI/PG/Tris buffer at 7.85:0.65:76.5:15 (w/w/w/w) complexed with 10 µM of siRNA, characterized as a mixture of cubic phase (diamond-type) and inverted hexagonal phase (after contact with excess water), showed sustained release for 7 days in vitro. In mice, in situ gel formation occurred after subcutaneous injection of the formulations, and the gels were degraded in 30 days. Initially a mild inflammatory process occurred in the tissue surrounding the gel; but after 14 days the tissue appeared normal. Taken together, this work demonstrates the rational development of an in situ gelling formulation for local release of siRNA.

Liquid crystalline phase nanodispersions enable skin delivery of siRNA.

The ability of small interfering RNAs (siRNAs) to potently but reversibly silence genes in vivo has made them particularly well suited as a new class of drugs that interfere with disease-causing or disease-promoting genes. However, the largest remaining hurdle for the widespread use of this technology in skin is the lack of an effective delivery system. The aim of the present study was to evaluate nanodispersed systems in liquid crystalline phases that deliver siRNA into the skin. The proposed systems present important properties for the delivery of macromolecules in a biological medium, as they are formed by substances that have absorption-enhancing and fusogenic effects; additionally, they facilitate entrapment by cellular membranes due to their nano-scale structure. The cationic polymer polyethylenimine (PEI) or the cationic lipid oleylamine (OAM) were added to monoolein (MO)-based systems in different concentrations, and after dispersion in aqueous medium, liquid crystalline phase nanodispersions were obtained and characterized by their physicochemical properties. Then, in vitro penetration studies using diffusion cell and pig ear skin were carried out to evaluate the effect of the nanodispersions on the skin penetration of siRNA; based on these results, the nanodispersions containing MO/OA/PEI/aqueous phase (8:2:5:85, w/w/w/w) and MO/OA/OAM/aqueous phase (8:2:2:88, w/w/w/w) were selected. These systems were investigated in vivo for skin penetration, skin irritation, and the ability to knockdown glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein levels in animal models. The results showed that the studied nanodispersions may represent a promising new non-viral vehicle and can be considered highly advantageous in the treatment of skin disorders; they were effective in optimizing the skin penetration of siRNA and reducing the levels of the model protein GAPDH without causing skin irritation.

Effects of low-level laser therapy on the oxidative metabolism and matrix proteins in the rat masseter muscle.

OBJECTIVE: This study aims to analyze the effects of low-level laser therapy (LLLT) on the oxidative activity and the expression/activity of metalloproteinases of the masseter muscle. BACKGROUND DATA: Currently in dentistry LLLT has been used on patients with muscular disorders, such as the temporomandibular disorders (TMDs) but its effect at the cellular level has not been fully elucidated. METHODS: Thirty male Wistar rats divided into 6 groups (n=5) received 10 laser irradiations (780 nm, 5 mmW, CW laser, illuminated area 0.04 cm(2), power density 125 mW/cm(2)), with different energy densities (group I-0; group II-0.5; group III-1.0; group IV-2.5; group V-5.0; and group VI-20 J/cm(2)). Muscles were processed for nicotinamide adenine dinucleotide diaphorase (NADH) and sucinate dehydrogenase (SDH) activities and zymography. The photomicrographs were evaluated by the point counting method using a test system and ImageJ software; and by the ANOVA statistical test. The proteinases' secretion/activity was qualitatively analyzed by zymography. RESULTS: LLLT significantly increased (p<0.05) masseter muscle oxidative metabolism shown by the increased area of intermediary fibers in the NADH (groups IV, V, and VI) and SDH (group V) reactions. The same metabolic pattern was observed among the groups in both reactions. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) zymography detected only the MMP-2 expression/activity for the untreated-control group (group I). The exposure to LLLT increased the activity of MPP-2 in group VI and the activity of MMP-9 in all groups exposed to different energy densities of laser irradiation (groups II, III, IV, V, and VI). CONCLUSIONS: Thus, LLLT stimulated the oxidative metabolism and the expression of matrix metalloproteinase (MMPs) of the masseter muscle, which may indicate a matrix remodeling process. However, group VI did not show the best results for oxidative metabolism, probably indicating that the dosage they were given was high for this protocol.