Intracellular trafficking and unpacking of siRNA/quantum dot-PEI complexes modified with and without cell penetrating peptide: confocal and flow cytometric FRET analysis.

Cationic quantum dots (QDs) were utilized to complex small interfering RNA (siRNA) for studying intracellular trafficking, unpacking, and gene silencing. Positively charged polyethylenimine (PEI) was covalently conjugated on the surface of QDs to complex with cyanine dye labeled vascular endothelial growth factor siRNA (cy5-VEGF siRNA) for the formation of nanosized polyelectrolyte complexes (PEC). Fluorescence resonance energy transfer (FRET) was achieved between cy5-VEGF siRNA and PEI conjugated QDs (QD625) in the complex. From confocal microscopic analysis, intracellular uptake and release of siRNA from the PEC were visualized as a function of incubation time. The extent of cy5-siRNA release from the PEC was quantitatively evaluated by flow cytometric analysis. In addition, PEI conjugated QDs were further modified with a protein transduction domain (PTD) from human transcriptional factor, Hph-1. The two siRNA/QD-PEI complexes with and without Hph-1 have shown markedly different intracellular uptake behaviors and unpacking kinetics of cy5-siRNA. However, they exhibited similar extent of VEGF gene knockout regardless of Hph-1, but showed much higher gene silencing efficiency than siRNA/PEI complexes. The present study demonstrates that PEI conjugated QDs can be utilized as a useful siRNA carrier to analyze intracellular trafficking and unpacking pathway as well as to effectively silence a target gene.

Analysis for the potential of polystyrene and TiO2 nanoparticles to induce skin irritation, phototoxicity, and sensitization.

The human skin equivalent model (HSEM) is well known as an attractive alternative model for evaluation of dermal toxicity. However, only limited data are available on the usefulness of a HSEM for nanotoxicity testing. This study was designed to investigate cutaneous toxicity of polystyrene and TiO2 nanoparticles using cultured keratinocytes, a HSEM, and an animal model. In addition, we also evaluated the skin sensitization potential of nanoparticles using a local lymph node assay with incorporation of BrdU. Findings from the present study indicate that polystyrene and TiO2 nanoparticles do not induce phototoxicity, acute cutaneous irritation, or skin sensitization. Results from evaluation of the HSEMs correspond well with those from animal models. Our findings suggest that the HSEM might be a useful alternative model for evaluation of dermal nanotoxicity.

Highly open porous biodegradable microcarriers: in vitro cultivation of chondrocytes for injectable delivery.

Injectable cell therapy would provide a patient-friendly procedure for treatment of degenerated or wounded tissue. Biodegradable injectable porous microspheres were fabricated to use as dual-purpose microcarriers for cell culture and injectable scaffold for tissue regeneration. Gas foaming in a water-in-oil-in-water double emulsion was performed for fabricating the well-interconnected porous microcarriers using poly(lactic-co-glycolic acid) (PLGA). The gas foaming conditions were finely tuned to control the structural and morphological characteristics. Porous microcarriers with a mean size of approximately 175 microm and an average pore diameter of approximately 29 microm were produced for cell cultivation and injectable delivery. To promote cell seeding, amine-functionalized porous microcarriers were prepared by blending amine-functionalized PLGA with unreacted PLGA. To assess the porous microcarriers for chondrocyte cultivation, bovine articular chondrocytes were seeded and cultured in vitro in spinner flasks for 4 weeks. Visualization and biochemical analyses of the microcarrier-cell constructs were performed to demonstrate cell proliferation and phenotypic expression. Quantification of deoxyribonucleic acid, glycosaminoglycan, and collagen content showed that much greater cell proliferation and expression of cartilage-specific phenotype were observed for cultures in the following order: amine-functionalized porous microcarriers, porous microcarriers, nonporous microcarriers, and monolayer culture.

Cationic solid lipid nanoparticles reconstituted from low density lipoprotein components for delivery of siRNA.

Cationic solid lipid nanoparticles (SLN), reconstituted from natural components of protein-free low-density lipoprotein, were used to deliver small interfering RNA (siRNA). The cationic SLN was prepared using a modified solvent-emulsification method. The composition was 45% (w/w) cholesteryl ester, 3% (w/w) triglyceride, 10% (w/w) cholesterol, 14% (w/w) dioleoylphosphatidylethanolamine (DOPE), and 28% (w/w) 3beta-[ N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-chol). The SLN had a mean diameter of 117+/-12 nm and a surface zeta potential value of +41.76+/-2.63 mV. A reducible conjugate of siRNA and polyethylene glycol (PEG) (siRNA-PEG) was anchored onto the surface of SLN via electrostatic interactions, resulting in stable complexes in buffer solution and in even 10% serum. Under an optimal weight ratio of DC-chol of SLN and siRNA-PEG conjugate, the complexes exhibited higher gene silencing efficiency of GFP and VEGF than that of polyethylenimine (PEI) 25K with showing much reduced cell cytotoxicity. Flow cytometry results also showed that siRNA-PEG/SLN complexes were efficiently taken up by cells. Surface-modified and reconstituted protein-free LDL mimicking SLN could be utilized as noncytotoxic, serum-stable, and highly effective carriers for delivery of siRNA.