carbocyanines and stearic-acid

The development of new nanoparticle formulations that are capable of high transfection efficiency without toxicity is essential to provide new tools for gene therapy. However, the issues of complex, poorly reproducible manufacturing methods, and low efficiencies during in vivo testing have prevented translation to the clinic. We have previously reported the use of cholesteryl oleate as a novel excipient for solid lipid nanoparticles (SLNs) for the development of highly efficient and nontoxic nucleic acid delivery carriers. Here, we performed an extensive characterization of this novel formulation to make the scale up under Good Manufacturing Practice (GMP) possible. We also describe the complete physicochemical and biological characterization of cholesteryl oleate-loaded SLNs to ensure the reproducibility of this formula and the preservation of its characteristics before and after the lyophilization process. We defined the best manufacturing method and studied the influence of some parameters on the obtained nanoparticles using the Quality by Design (ICH Q8) guideline to obtain cholesteryl oleate-loaded SLNs that remain stable during storage and guarantee in vitro nucleic acid delivery efficacy. Our results indicate that this improved formulation is suitable for gene therapy with the possibility of scale-up the manufacturing of nanoparticles under GMP conditions.

Topics: Amines; Carbocyanines; Cations; Cholesterol Esters; Factor Analysis, Statistical; Fluorescent Dyes; Gene Transfer Techniques; HeLa Cells; Humans; Microscopy, Fluorescence; Nanoparticles; Particle Size; Plasmids; Poloxamer; RNA, Small Interfering; Stearic Acids; Transfection

Dendritic core-multi shell (CMS) particles are polymer based systems consisting of a dendritic polar polyglycerol polymer core surrounded by a two-layer shell of nonpolar C18 alkyl chains and hydrophilic polyethylene glycol. Belonging to nanotransport systems (NTS) they allow the transport and storage of molecules with different chemical characters. Their amphipihilic character CMS-NTS permits good solubility in aqueous and organic solutions. We showed by multifrequency electron paramagnetic resonance (EPR) spectroscopy that spin-labeled 5-doxyl stearic acid (5DSA) can be loaded into the CMS-NTS. Furthermore, the release of 5DSA from the carrier into the stratum corneum of porcine skin was monitored ex vivo by EPR spectroscopy. Additionally, the penetration of the CMS-NTS into the skin was analyzed by fluorescence microscopy using indocarbocyanine (ICC) covalently bound to the nanocarrier. Thereby, no transport into the viable skin was observed, whereas the CMS-NTS had penetrated into the hair follicles down to a depth of 340 μm ± 82 μm. Thus, it could be shown that the combined application of fluorescence microscopy and multi-frequency EPR spectroscopy can be an efficient tool for investigating the loading of spin labeled drugs to nanocarrier systems, drug release and penetration into the skin as well as the localization of the NTS in the skin.

Topics: Administration, Cutaneous; Animals; Carbocyanines; Dendrimers; Drug Carriers; Electron Spin Resonance Spectroscopy; Fluorescent Dyes; Glycerol; Hair Follicle; In Vitro Techniques; Microscopy, Fluorescence; Nanostructures; Polyethylene Glycols; Polymers; Skin; Skin Absorption; Stearic Acids; Swine

We introduce a fast and simple one-step method, a variation of the methods of Barrett and Campbell and Bartlett, to synthesize monodisperse fluorescent particles that can be dispersed in organic solvents and have long excitation (649 nm) and emission wavelengths (679 nm). A lipophilic fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate, is directly incorporated into PMMA particles through dispersion polymerization. A poly(hydroxystearic acid) graft (poly) methyl methacrylate (MM) and methacrylic acid (MA) copolymer is used as a stabilizer to prevent the particles from aggregating and flocculating in the nonaqueous solvents. The fluorescent PMMA particles are very uniform in size, bleach at very low rate, and behave like hard spheres in their ordering on substrates. One important achievement in our synthesis protocol is that we are able to produce particles of a desired size by choosing the composition of the reactants according to a predetermined relationship between particle size and composition of reactants. In addition, the effects of fluorescent dye and polar solvent (ethanol) on the formation and size of particles are discussed.

Topics: Biocompatible Materials; Carbocyanines; Excipients; Fluorescent Dyes; Materials Testing; Microscopy, Fluorescence; Organic Chemicals; Particle Size; Polymethacrylic Acids; Solubility; Solvents; Stearic Acids