Targeting cellular gaps using Janus nanoparticles containing cationic polymers and surfactant lipids.

Since nanoparticles are taken up into cells by endocytosis, phagocytosis, or pinocytosis, they have been studied as intracellular drug carriers. Janus particles have an anisotropic structure composed of two or more distinct domains and have been proposed for use in various applications, including use as imaging agents or nanosensors. This study aimed to clarify the influence of the type of nanoparticles on their distribution in a human Caucasian colon adenocarcinoma (Caco-2) cell monolayer. We fabricated Janus and conventional spherical nanoparticles composed of pharmaceutically applicable ingredients. Janus and spherical nanoparticles composed of a cationic polymer and surfactant lipids were prepared by controlling the solvent removal pattern from the oil phase in the solvent removal process using the solvent evaporation and solvent diffusion methods. The distribution of nanoparticles in the Caco-2 cell monolayer was then evaluated using confocal laser microscopy. The mean hydrodynamic size of the fabricated Janus nanoparticles was 119.2 ± 4.6 nm. Distribution analysis using Caco-2 cells suggested that Janus nanoparticles were localized around the adherens junctions located just below the tight junction. Clear localization was not observed in non-Janus nanoparticles with the same composition. The clear localization of the Janus nanoparticles around the adherens junction may be due to their positive charge and asymmetric structure. Our results suggest the considerable potential for the development of nanoparticulate drug carriers to target cellular gaps.

ATP13A2 modifies mitochondrial localization of overexpressed TOM20 to autolysosomal pathway.

Mutations in ATP13A2 cause Kufor-Rakeb Syndrome (KRS), a juvenile form of Parkinson's Disease (PD). The gene product belongs to a diverse family of ion pumps and mediates polyamine influx from lysosomal lumen. While the biochemical and structural studies highlight its unique mechanics, how PD pathology is linked to ATP13A2 function remains unclear. Here we report that localization of overexpressed TOM20, a mitochondrial outer-membrane protein, is significantly altered upon ATP13A2 expression to partially merge with lysosome. Using Halo-fused version of ATP13A2, ATP13A2 was identified in lysosome and autophagosome. Upon ATP13A2 co-expression, overexpressed TOM20 was found not only in mitochondria but also within ATP13A2-containing autolysosome. This modification of TOM20 localization was inhibited by adding 1-methyl-4-phenylpyridinium (MPP+) and not accompanied with mitophagy induction. We suggest that ATP13A2 may participate in the control of overexpressed proteins targeted to mitochondrial outer-membrane.

A novel preparation method for PLGA microspheres using non-halogenated solvents.

We investigated a new preparation method for microspheres based on an Oil/Water type emulsion solvent evaporation method using non-halogenated solvents. This method is based on phase separation between acetone and aqueous glycerol. For the preparation of microspheres by this method, a solution of poly(DL-lactic-co-glycolic acid) (PLGA) in acetone and aqueous glycerol containing poly(vinyl alcohol) were used asthe dispersed and continuous phases in the emulsification process, respectively. Vitamin B12 was used as the model drug. The formation of PLGA microspheres was observed above 60% glycerol in the continuous phase. The yield and encapsulation efficiency of the PLGA microspheres was about 80%, which was the maximum yield obtained with 70% glycerol. The release of vitamin B12 lasted for three weeks.

Enhancement of intestinal absorption of poorly absorbed hydrophilic compounds by simultaneous use of mucolytic agent and non-ionic surfactant.

The effect of co-administration of a mucolytic agent with a penetration enhancer was assessed on the intestinal absorption of poorly absorbed hydrophilic compounds. Fluorescein isothiocyanate-labeled dextran with average molecular weight of ca. 4.4 kDa (FD-4) was used as a model compound, and N-acetylcysteine (NAC) was used as a mucolytic agent. Sodium caprate (C10), tartaric acid (TA), sodium taurodeoxycholate (TDC), sodium dodecyl sulfate (SDS), p-t-octyl phenol polyoxyethylene-9.5 (Triton X-100, TX-100) were selected as penetration enhancers with different mechanisms of action. Various dosing solutions containing a penetration enhancer in the absence or in the presence of NAC were directly administered into the exposed rat jejunum, and the bioavailability of FD-4 up to 2 h was determined. The extent of improvement by co-administration was highly dependent on the penetration enhancer species applied. The observed enhancement was thought to result from the mucolytic activity of NAC, which can reduce the mucus viscosity and facilitate the penetration of FD-4 to mucosal membrane. Among the combinations tested, the simultaneous administration of NAC and TX-100 provided the highest enhancement (22.5-fold) of intestinal FD-4 absorption compared to the control. Although the detailed mechanism for the observed drastic improvement is unclear, one possible reason was thought to be due to the improved diffusivity of TX-100 micellar system in the mucus layer. All these results suggest that the combination of a mucolytic agent and a non-ionic surfactant may have potential as an enhancing system for peroral delivery of poorly absorbed hydrophilic compounds like protein and peptide drugs.

In vitro study of the functional expression of organic anion transporting polypeptide 3 at rat choroid plexus epithelial cells and its involvement in the cerebrospinal fluid-to-blood transport of estrone-3-sulfate.

The cerebrospinal fluid-to-blood efflux transport of estrone-3-sulfate (E(1)S) via the blood-cerebrospinal fluid barrier (BCSFB) may play an important role in regulating E(1)S levels in the brain. Here, we investigated the efflux transport of E(1)S at the BCSFB using conditionally immortalized rat choroid plexus epithelial cells (TR-CSFB) and identified the responsible transporter. The [(3)H]E(1)S uptake by TR-CSFB cells was composed of saturable and nonsaturable components, and the K(m) and V(max) values of the saturable component were determined to be 16.8 +/- 5.1 microM and 12.3 +/- 2.3 pmol/min/mg of protein, respectively. [(3)H]E(1)S uptake was inhibited by probenecid, cholate, taurocholate, sulfobromophthalein, dehydroepiandrosterone sulfate, triiodothyronine, thyroxin, and digoxin but not by p-aminohippuric acid, gamma-aminobutyric acid, or methotrexate, suggesting the involvement of organic anion transporting polypeptide (oatp) in the uptake. Reverse transcription-polymerase chain reaction analysis revealed that oatp3 was expressed in TR-CSFB cells and isolated rat choroid plexus, although oatp1 was not detected in either. Xenopus laevis oocytes expressing oatp3 exhibited [(3)H]E(1)S uptake activity with a K(m) of 8.09 +/- 2.83 microM and V(max) of 8.02 +/- 0.87 pmol/h/oocyte. Moreover, oatp3 is localized at the brush-border membrane of choroid plexus epithelial cells. These results suggest that oatp3 is involved in the E(1)S efflux transport at the BCSFB.