Polyoxazolines based lipid nanocapsules for topical delivery of antioxidants.

Nano-sized lipid formulations offer a great potential for topical delivery of active compounds to treat and prevent human skin damages. Of particular importance is the high loading of hydrophobic molecules, the long-term stability and the auspicious penetration capacity especially reached when using lipid nanocapsules (LNC). Unfortunately, their formation currently relies on a phase inversion process that only operates when using a poly(ethylene glycol) (PEG) based surfactant belonging to the controversial PEG family that was subject of clinical awareness. The present study proposes an alternative to this overused polymer in formulations by designing LNC made of harmless amphiphilic polyoxazolines (POx). Implementing a short sonication step in the process allowed well-defined spherical nanoparticles of ~30 nm to be obtained. The structure of the so called LNC POx was composed of an oily core surrounded by a rigid shell of phospholipids and POx, which ensures a high stability over time, temperature, centrifugation and freezing. Encapsulation of the natural quercetin antioxidant led to a drug loading three times higher than for LNC constituted of PEG (LNC PEG). The antioxidant activity of loaded LNC POx was tested on mice fibroblasts and human keratinocytes after exposure to free radicals from peroxides and UVB irradiation, respectively. The radical scavenging capacity of quercetin loaded in the LNC POx was preserved and even slightly enhanced compared to LNC PEG, highlighting the POx value in nanoformulations.

Brain engraftment of autologous macrophages transduced with a lentiviral flap vector: an approach to complement brain dysfunctions.

Transplantation of ex vivo gene-corrected autologous cells represents an attractive therapeutic approach for brain diseases. Among the cells of the central nervous system, brain macrophages are promising candidates due to their role in tissue homeostasis and their implication in several neurological diseases. Up to now, gene transfer into macrophages has proven difficult by most currently available gene delivery methods. We describe herein, an efficient transduction of rat bone marrow-derived and brain macrophages with an HIV-1-derived vector containing a central DNA flap and encoding the GFP reporter gene (TRIP-DeltaU3-GFP). In primary cultures of macrophages our results show that more than 90% of the cells were transduced by the TRIP vector and that GFP expression remained stable for 1 month without cytopathic effect. In vivo, transplants of transduced macrophages into the striatum of adult rats exhibited long-term expression of GFP up to 3 months. Transduced macrophages were observed around the brain injection site and exhibited the brain macrophage/microglia phenotype. There was no significant sign of astrogliosis around the graft. These results confirm the potential of lentiviral vectors for efficient and stable ex vivo transduction of macrophages. Moreover, transduced autologous macrophages appear as a valuable vehicle for long-term and localized gene expression into the brain.

Preferential transduction of neurons by canine adenovirus vectors and their efficient retrograde transport in vivo.

In the central nervous system (CNS), there are innate obstacles to the modification of neurons: their relative low abundance versus glia and oligodendrocytes, the inaccessibility of certain target populations, and the volume one can inject safely. Our aim in this study was to characterize the in vivo efficacy of a novel viral vector derived from a canine adenovirus (CAV-2). Here we show that CAV-2 preferentially transduced i) rat olfactory sensory neurons; ii) rodent CNS neurons in vitro and in vivo; and, more clinically relevant, iii) neurons in organotypic slices of human cortical brain. CAV-2 also showed a high disposition for retrograde axonal transport in vivo. We examined the molecular basis of neuronal targeting by CAV-2 and suggest that due to CAR (coxsackie adenovirus receptor) expression on neuronal cells-and not oligodendrocytes, glia, myofibers, and nasal epithelial cells-CAV-2 vectors transduced neurons preferentially in these diverse tissues.

Induction of early transcription in one-cell mouse embryos by microinjection of the nonhistone chromosomal protein HMG-I.

In the mouse embryo, the onset of zygotic transcription occurs at the end of the first cell cycle, upon completion of DNA replication. We show that the nonhistone chromosomal protein HMG-I, whose translocation into the pronuclei of one-cell embryos is linked to this first round of DNA synthesis, plays a critical role in the activation of zygotic transcription. Indeed, microinjection of purified HMG-I results in a higher nuclear accumulation of the protein and triggers an earlier activation of zygotic transcription, an effect which is abolished by the preincubation of the protein with a specific antibody directed against its AT-hook DNA-binding motifs. Significantly, microinjection of this antibody also prevents the normal onset of transcription in the embryo, suggesting that endogenous HMG-I is similarly involved in this process. Finally, microinjection of the exogenous protein modifies chromatin structure as measured by in situ accessibility to DNase I. We propose that general chromosomal architectural factors such as HMG-I can modulate the accessibility of chromatin to specialized regulatory factors, thereby promoting a transcriptionally competent state.

A somatic gene transfer approach using recombinant fusion proteins to map muscle-motoneuron projections in Xenopus spinal cord.

A combination of somatic gene transfer with fusion protein technology has been developed, thus providing an innovative means of mapping muscle-motoneuronal connections in Xenopus tadpole spinal cord. We analyzed whether a neuronal tracer created by the fusion of the LacZ gene to the tetanus toxin C fragment (LacZ-TTC) could be produced from plasmid DNA injected into muscle, and whether it could be released and undergo retrograde transport into motoneurons. Plasmids encoding various fusion protein constructions, with or without a signal peptide, were injected into dorsal or caudal muscles of premetamorphic tadpoles. The marker was produced in the muscle at constantly high levels. At one month post-injection, the fusion protein passed the neuromuscular junction and underwent retrograde transport into motoneurons. Transfer into motoneurons was seen for every animal injected, emphasizing the high reproducibility and efficiency of the process. No uptake of beta-gal protein into motoneurons was observed in the absence of the TTC fragment. Furthermore, no enhancement was obtained by adding a signal peptide. These results provide the first demonstration of the synthesis and transport of a TTC fusion protein produced directly from exogenous DNA in a vertebrate system.

Use of radio-frequency-pulse surface acoustic wave profilometry for passband engineering of acousto-optic tunable filters.

One can determine the acoustic profile of an acousto-optic tunable filter (AOTF) either by measuring the intensity of a laser diffracted off the surface of an AOTF by the surface acoustic wave (SAW) or by measuring the optical output power of an AOTF as a short burst of rf energy is launched down the device. This rf-burst technique (also called pulse probing) is compared with the established optical diffraction SAW profilometry technique. We demonstrate the usefulness of rf-burst profilometry to measure the acousto-optic coupling as a function of position along an AOTF interaction region. Such a probe can be used to make adjustments to the acousto-optic coupling in real time to achieve passband shape modification in complex AOTF structures.