Interaction of poly(butylcyanoacrylate) nanoparticles with the blood-brain barrier in vivo and in vitro.

Poly(butylcyanoacrylate) nanoparticles were produced by emulsion polymerisation and used either uncoated or overcoated with polysorbate 80 (Tween 80). [3H]-dalargin bound to nanoparticles overcoated with polysorbate 80 or in the form of saline solution was injected into mice and the brain concentrations of radioactivity determined. Statistically significant, three-fold higher brain concentrations with the nanoparticle preparations were obtained after 45 minutes, the time of greatest pharmacological response assessed as analgesia in previous experiments. In addition the brain inulin spaces in rats and the uptake of fluoresceine isothiocyanate labelled nanoparticles in immortalised rat cerebral endothelial cells, (RBE4) were measured. The inulin spaces after i.v. injection of polysorbate 80-coated nanoparticles were significantly increased by 1% compared to controls. This is interpreted as indicating that there is no large scale opening of the tight junctions of the brain endothelium by the polysorbate 80-coated nanoparticles. In in vitro experiments endocytic uptake of fluorescent nanoparticles by RBE4 cells was only observed after polysorbate 80-overcoating, not with uncoated particles. These results further support the hypothesis that the mechanism of blood-brain barrier transport of drugs by polysorbate 80-coated nanoparticles is one of endocytosis followed by possible transcytosis. The experiments were conducted in several laboratories as part of an EEC/INTAS collaborative program. For various procedural and regulatory reasons this necessitated the use of both rats and mice as experimental animals. The brain endothelial cell line used for the in vitro studies is the rat RBE4.

Polysorbate-80 coating enhances uptake of polybutylcyanoacrylate (PBCA)-nanoparticles by human and bovine primary brain capillary endothelial cells.

Certain drugs such as dalargin, loperamide or tubocurarine are not transported across the blood-brain barrier (BBB) and therefore exhibit no effects on the central nervous system. However, effects on the central nervous system can be observed when these drugs are loaded onto polybutylcyanoacrylate (PBCA)-nanoparticles and coated with polysorbate 80. The mechanism by which these complexed nanoparticles cross the BBB and exhibit their effects has not been elucidated. Cultured microvessel brain endothelial cells of human and bovine origin were used as an in vitro model for the BBB to gain further insight into the mechanism of uptake of nanoparticles. With cells from these species we were able to show that polysorbate 80-coated nanoparticles were taken up by brain endothelial cells much more rapidly and in significantly higher amounts (20-fold) than uncoated nanoparticles. The process of uptake was followed by fluorescence and confocal laser scanning microscopy. The results demonstrate that the nanoparticles are taken up by cells and that this uptake occurs via an endocytotic mechanism.

Circadian phase-dependent antinociceptive reaction in mice determined by the hot-plate test and the tail-flick test after intravenous injection of dalargin-loaded nanoparticles.

Peptides normally do not cross the blood-brain barrier (BBB). Previously, it has been shown that the hexapeptide enkephalin analogue dalargin with polysorbate-80-coated nanoparticles (DAL/NP) can be transported across the BBB and is able to exhibit an antinociceptive effect in mice. In the present study, the circadian time and dose dependencies of the antinociceptive effect of different dalargin preparations were investigated. The active preparation (DAL/NP, 5 mg/kg, 10 mg/kg), as well as a dalargin solution in phosphate buffered saline (DAL/SOL, 10 mg/kg) were injected intravenously to groups of 10-12 inbred DBA/2 mice at 12 different circadian times; mice were synchronized to a light-dark (LD) 12:12 regimen. The antinociceptive effect was determined 15 minutes postinjection by the hot-plate test. Experiments with DAL/NP were repeated using the tail-flick test system at two selected times (08:00 and 20:00) to test for dose dependency (2.5, 5, 7.5, 10 mg/kg). Hot-plate latencies were rhythmic under baseline and after DAL/SOL, with acrophases in the dark phase; DAL/SOL did not influence latency time. In contrast, DAL/NP significantly increased reaction time dose dependently; the maximal possible effect was rhythmic with the 10 mg/kg preparation, with a peak effect in the early light phase. Results were confirmed by the tail-flick test. The experiments demonstrate that an enkephalin analogue coated with nanoparticles can easily cross the BBB and is able to display a dose- and time-dependent antinociceptive effect.

Significant entry of tubocurarine into the brain of rats by adsorption to polysorbate 80-coated polybutylcyanoacrylate nanoparticles: an in situ brain perfusion study.

The possibility of using polysorbate 80-coated polybutylcyanoacrylate nanoparticles to deliver low molecular polar hydrophilic drugs to the CNS has been studied. Tubocurarine (a quaternary ammonium salt) does not penetrate the normal intact blood-brain barrier. However, the injection of this drug directly into the cerebral ventricles of the brain provokes the development of epileptiform seizures as assessed by electroencephalogram (EEG). An in situ perfused rat brain technique was used as an experimental technique together with a simultaneous recording of the EEG. Nanoparticles were prepared by butylcyanoacrylate polymerization in an acidic medium. Fifteen minutes after the introduction of tubocurarine-loaded polysorbate 80-coated nanoparticles into the perfusate, epileptiform spikes in the EEG appeared. Intraventricular injection of tubocurarine caused the appearance of the EEG seizures 5 min after administration. Neither tubocurarine solution nor tubocurarine-loaded nanoparticles without polysorbate 80 or a mixture of polysorbate 80 and tubocurarine were able to influence the EEG. Thus only the loading of tubocurarine onto the polysorbate 80-coated nanoparticles appears to enable the transport of this quaternary ammonium compound through the blood-brain barrier.

Apoplastic antioxidants as decisive elimination factors within the uptake process of nitrogen dioxide into leaf tissues.

Uptake of NO2 via stomata and its elimination in the mesophyll are investigated by use of mathematical models. Systems of differential equations describe simultaneous diffusion and reaction of the chemical species taken into account. The common hypothesis, postulating the disproportionation reaction of NO2 being the predominant path for the transformation of NO2 into the cellular nitrate and nitrite pools, could be false because of its failure to reproduce the measured sorption characteristics. If however the reduction of the pro-oxidative NO2 by apoplastic ascorbate is taken into account the calculated uptake rates and the effects of changes in immission concentration and vegetational characteristics fit well within corresponding experimental results. The onset of acute injury in case of a fall in apoplastic ascorbate concentration below a critical level is postulated.