Nanoparticulate Transport of Oximes over an In Vitro Blood-Brain Barrier Model

Background: Due to the use of organophosphates (OP) as pesticides and the availability of OP-type nerve agents, an effective medical treatment for OP poisonings is still a challenging problem. The acute toxicity of an OP poisoning is mainly due to the inhibition of acetylcholinesterase (AChE) in the peripheral and central nervous systems (CNS). This results in an increase in the synaptic concentration of the neurotransmitter acetylcholine, overstimulation of cholinergic receptors and disorder of numerous body functions up to death. The standard treatment of OP poisoning includes a combination of a muscarinic antagonist and an AChE reactivator (oxime). However, these oximes can not cross the blood-brain barrier (BBB) sufficiently. Therefore, new strategies are needed to transport oximes over the BBB. Methodology/Principal Findings: In this study, we combined different oximes (obidoxime dichloride and two different HI 6 salts, HI 6 dichloride monohydrate and HI 6 dimethanesulfonate) with human serum albumin nanoparticles and could show an oxime transport over an in vitro BBB model. In general, the nanoparticulate transported oximes achieved a better reactivation of OP-inhibited AChE than free oximes. Conclusions/Significance: With these nanoparticles, for the first time, a tool exists that could enable a transport of oximes over the BBB. This is very important for survival after severe OP intoxication. Therefore, these nanoparticulate formulation

Human serum Albumin-based nanoparticle-mediated in vitro gene delivery

The genetic treatment of neurodegenerative diseases still remains a challenging task since many approaches fail to deliver the therapeutic material in relevant concentrations into the brain. As viral vectors comprise the risk of immune and inflammatory responses, human serum albumin (HSA) nanoparticles were found to represent a safer and more convenient alternative. Their ability to cross the blood-brain barrier (BBB) and deliver drugs into the brain in order to enhance gene-based therapy has been previously demonstrated. The present study deals with the development of pGL3-PEI-coated HSA nanoparticles and subsequent in vitro testing in cerebellar granular and HeLa cells. The luciferase control vector pGL3 was chosen as reporter plasmid encoding for the firefly luciferase protein, linear polyethylenimine (22 kDa) as endosomolytic agent for enhancing the cells’ transfection. Studies on particle characteristics, their cellular uptake into aforementioned cell lines and on subcellular localisation, and transfection efficiency in the cerebellar cells proved the feasibility of nanoparticle-based gene delivery

<i>In-vitro</i> expression levels of luciferase reporter protein.

<p>Cb cells were transfected with pGL3-PEI-coated nanoparticles. The graph shows the percentage of positive wells showing luminescence on day 2, 3, 4 after washing the nanoparticles off. Well values higher than negative control average + 3x standard deviation, were considered positive. Results are shown as mean ± S.E.M. (n = 48).</p

Particle characteristics of pGL3-PEI-coated nanoparticles, unmodified HSA nanoparticles, and pGL3-PEI complexes.

<p>Particle sizes [nm] (<b>A</b>) and surface charge [mV] (<b>B</b>) of unmodified HSA nanoparticles, pGL3-PEI complexes, and pGL3-PEI-coated HSA nanoparticles. Results are shown as mean ± S.E.M. (n = 3). (<b>C</b>) Scanning electron microscopy picture of pGL3-PEI coated HSA nanoparticles. Scale bar 700 nm.</p

Degradation of pGL3-PEI-coated nanoparticles.

<p><b>A:</b> Dot blot immunoanalysis was used to evaluate the internalised HSA amount in Cb cells treated with pGL3-PEI-coated nanoparticles The HSA signal was related to the signal of the negative control (horizontal line). <b>B:</b> Similar conditions were used to evaluate the chemical degradation of pGL3-PEI-coated HSA nanoparticles with proteinase K. The HSA signal was related to the signal of non-degraded nanoparticles (horizontal line). Results are shown as mean ± S.E.M. (n = 3).</p

Uptake of HSA nanoparticles and pGL3-PEI-coated HSA nanoparticles in cerebellar granule cells.

<p><b>A:</b> Flow cytometry was used to determine the autofluorescence of the HSA nanoparticles in Cb cells. Nanoparticle uptake into the cells increased with higher concentrations of used nanoparticles (0.1 mg/ml to 1.0 mg/ml) and with incubation time (18 h to 72 h). <b>B:</b> Temperature dependency of nanoparticles uptake at 4°C, 33°C and 39°C. <b>C:</b> Comparison between unmodified and pGL3-PEI-coated HSA nanoparticle uptake in Cb cells. Results are shown as mean ± S.E.M. (n = 3). <b>D:</b> Confocal microscopy images represent the uptake of nanoparticles in Cb cells at different concentrations. 0.1 mg/ml (1), 0.25 mg/ml (2), 0.5 mg/ml (3), 1 mg/ml (4). Scale bar 100 µm.</p

Cellular uptake and intracellular distribution of the nanoparticles studied by CLSM.

<p>bEnd3 cells were cultured on collagen IV-coated glass slides and were treated with a) PEGylated HI 6 dichloride monohydrate-loaded nanoparticles or b) ApoE-modified HI 6 dichloride monohydrate-loaded nanoparticles for 4 h at 37°C. The green autofluorescence of the nanoparticles was used for detection. Red: cytosol stained with CellTracker™ Red CMTPX, blue: nucleus stained with DAPI. Pictures were taken within inner sections of the cells.</p

Transport study of adsorptively HI 6 dimethanesulfonate-loaded nanoparticles on an <i>in vitro</i> BBB model.

<p>*Mann-Whitney U Test: significant difference between NP-ApoE and free HI 6-DMS (P<0.05).</p

Transport study of adsorptively obidoxime-loaded nanoparticles on an <i>in vitro</i> BBB model.

<p>Transport study of adsorptively obidoxime-loaded nanoparticles on an <i>in vitro</i> BBB model.</p