Resveratrol-Loaded Lipid Nanocarriers Are Internalized By Endocytosis in Yeast.

Different positive pharmacological effects have been attributed to the natural product resveratrol (RSV), including antioxidant, antiaging, and cancer chemopreventive properties. However, its low bioavailability and rapid metabolic degradation has led to the suspicion that many of the biological activities of this compound observed in vitro may not be attainable in humans. To improve its bioavailability and pharmacokinetic profile, attempts have been made to encapsulate RSV into lipid-based nanocarrier systems. Here, the dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) liposomal system (1:2) loaded with RSV revealed appropriate characteristics for drug release purposes: reduced size for cellular uptake (157 ± 23 nm), stability up to 80 days, positive surface charge (ζ ≈ +40 mV), and a controlled biphasic release of RSV from the lipid nanocarriers over a period of almost 50 h at pH 5.0 and 7.4. Moreover, the encapsulation efficiency of the nanocarrier ranged from 70% to 92% and its RSV loading capacity from 9% to 14%, when [RSV] was between 100 and 200 µM. The partition coefficient ( Kp) of RSV between lipid and aqueous phase was log Kp = 3.37 ± 0.10, suggesting moderate to high lipophilicity of this natural compound and reinforcing the lipid nanocarriers' suitability for RSV incorporation. The thermodynamic parameters of RSV partitioning in the lipid nanocarriers at 37 °C (Δ H = 43.76 ± 5.68 kJ mol-1; Δ S = 0.20 ± 0.005 kJ mol-1; and Δ G = -18.46 ± 3.48 kJ mol-1) reflected the spontaneity of the process and the establishment of hydrophobic interactions. The cellular uptake mechanism of the RSV-loaded nanocarriers labeled with the lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was studied in the eukaryotic model system Saccharomyces cerevisiae. Thirty minutes after incubation, yeast cells readily internalized nanocarriers and the spots of blue fluorescence of DPH clustered around the central vacuole in lipid droplets colocalized with the green fluorescence of the lipophilic endocytosis probe FM1-43. Subsequent studies with the endocytosis defective yeast deletion mutant ( end3Δ) and with the endocytosis inhibitor methyl-ß-cyclodextrin supported the involvement of an endocytic pathway. This novel nanotechnology approach opens good perspectives for medical applications.

Structural dynamics and physicochemical properties of pDNA/DODAB:MO lipoplexes: effect of pH and anionic lipids in inverted non-lamellar phases versus lamellar phases.

Dioctadecyldimethylammonium bromide (DODAB):Monoolein (MO) lipoplexes have mainly been studied within the range of high molar ratios of DODAB, with noticeable transfection efficiencies in the Human Embryonic Kidney (HEK, a.k.a. 293T) cell line. In this work, we intend to study the effect of high MO content on the structure and physicochemical properties of pDNA/DODAB:MO lipoplexes to achieve some correlation with their transfection efficiency. Static/Dynamic Light Scattering and Cryo-TEM imaging were used to characterize the size/morphology of DNA/DODAB:MO lipoplexes at different DODAB:MO contents (2:1, 1:1, 1:2) and charge ratios (CRs) (+/-). Nile Red fluorescence emission was performed to detect changes in microviscosity, hydration and polarity of DNA/DODAB:MO systems. Lipoplexes stability at physiological pH values and in the presence of anionic lipids was evaluated by Förster Resonance Energy Transfer (FRET). Physicochemical/structural data were complemented with transfection studies in HEK cells using the ß-galactosidase reporter gene activity assay. This work reports the coexistence of multilamellar and non-lamellar inverted phases in MO-richer lipoplexes (DODAB:MO 1:2 and 1:4), leading to transfection efficiencies comparable to those of multilamellar (DODAB-richer) lipoplexes, but at higher charge ratios [CR (+/-)=6.0] and without dose-effect response. These results may be related to the structural changes of lipoplexes promoted by high MO content.

DODAB:monoolein-based lipoplexes as non-viral vectors for transfection of mammalian cells.

DNA/Cationic liposome complexes (lipoplexes) have been widely used as non-viral vectors for transfection. Neutral lipids in liposomal formulation are determinant for transfection efficiency using these vectors. In this work, we studied the potential of monoolein (MO) as helper lipid for cellular transfection. Lipoplexes composed of pDNA and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at different molar ratios (4:1, 2:1 and 1:1) and at different cationic lipid/DNA ratios were investigated. The physicochemical properties of the lipoplexes (size, charge and structure), were studied by Dynamic Light Scattering (DLS), Zeta Potential (ζ) and cryo-transmission electron microscopy (cryo-TEM). The effect of MO on pDNA condensation and the effect of heparin and heparan sulphate on the percentage of pDNA release from the lipoplexes were also studied by Ethidium Bromide (EtBr) exclusion assays and electrophoresis. Cytotoxicity and transfection efficiency of these lipoplexes were evaluated using 293T cells and compared with the golden standard helper lipids 1,2-dioleoyl-sn-glycero-3-hosphoethanolamine (DOPE) and cholesterol (Chol) as well as with a commercial transfection agent (Lipofectamine™ LTX). The internalization of transfected fluorescently-labeled pDNA was also visualized using the same cell line. The results demonstrate that the presence of MO not only increases pDNA compactation efficiency, but also affects the physicochemical properties of the lipoplexes, which can interfere with lipoplex-cell interactions. The DODAB:MO formulations tested showed little toxicity and successfully mediated in vitro cell transfection. These results were supported by fluorescence microscopy studies, which illustrated that lipoplexes were able to access the cytosol and deliver pDNA to the nucleus. DODAB:MO-based lipoplexes were thus validated as non-toxic, efficient lipofection vectors for genetic modification of mammalian cells. Understanding the relation between structure and activity of MO-based lipoplexes will further strengthen the development of these novel delivery systems.

Lipid-based Nanocarriers for siRNA Delivery: Challenges, Strategies and the Lessons Learned from the DODAX: MO Liposomal System.

The possibility of using the RNA interference (RNAi) mechanisms in gene therapy was one of the scientific breakthroughs of the last century. Despite the extraordinary therapeutic potential of this approach, the need for an efficient gene carrier is hampering the translation of the RNAi technology to the clinical setting. Although a diversity of nanocarriers has been described, liposomes continue to be one of the most attractive siRNA vehicles due to their relatively low toxicity, facilitated siRNA complexation, high transfection efficiency and enhanced pharmacokinetic properties. This review focuses on RNAi as a therapeutic approach, the challenges to its application, namely the nucleic acids' delivery process, and current strategies to improve therapeutic efficacy. Additionally, lipid-based nanocarriers are described, and lessons learned from the relation between biophysical properties and biological performance of the dioctadecyldimethylammonium:monoolein (DODAX: MO) system are explored. Liposomes show great potential as siRNA delivery systems, being safe nanocarriers to protect nucleic acids in circulation, extend their half-life time, target specific cells and reduce off-target effects. Nevertheless, several issues related to delivery must be overcome before RNAi therapies reach their full potential, namely target-cell specificity and endosomal escape. Understanding the relationship between biophysical properties and biological performance is an essential step in the gene therapy field.