Crosslinked nanocarriers based upon poly(ethylene imine) for systemic plasmid delivery: in vitro characterization and in vivo studies in mice.

Crosslinked poly(ethylene imine) (PEI) polyplexes for intracellular DNA release were generated using a low molecular weight crosslinking reagent, Dithiobis(succinimidyl propionate) (DSP). Disulfide bonds of the crosslinked polyplexes were susceptible to intracellular redox conditions and DNA release was observed using an ethidium bromide exclusion assay and dynamic light scattering. Transfection experiments were performed to elucidate the effect of extra- and intracellular redox conditions. Pharmacokinetics and organ accumulation of uncrosslinked and crosslinked polyplexes were compared and gene expression patterns were measured in mice 24 h after intravenous injection. Crosslinked PEI and plasmid DNA formed stable polyplexes in a size range of 100-300 nm, with zeta potentials between +16.4 and +26.1 mV. DNA release occurred after cleavage of the disulfide bonds. Cell culture experiments under reducing conditions as well as with glutathione loaded cells confirmed the proposed intracellular activation. A significant influence of the intracellular glutathione status on the transfection efficiency was observed. Pharmacokinetic profiles of crosslinked PEI/DNA polyplexes in mice after intravenous administration showed higher blood levels for crosslinked polyplexes. These polyplexes accumulated mainly in the liver and the lungs. In vivo transfection data revealed significantly reduced (unwanted) lung transfection while liver transfection predominated. These studies suggest that crosslinked polyplexes are more stable in circulation and retain their transfection efficiency after intravenous administration.

PEGylation of poly(ethylene imine) affects stability of complexes with plasmid DNA under in vivo conditions in a dose-dependent manner after intravenous injection into mice.

The influence of PEGylation on polyplex stability from poly(ethylene imine), PEI, and plasmid DNA was investigated both in vitro and after intravenous administration in mice. Polyplexes were characterized with respect to particle size (dynamic light scattering), zeta-potential (laser Doppler anemometry), and morphology (atomic force microscopy). Pharmacokinetics and organ accumulation of both polymers and pDNA were investigated using 125I and 32P radioactive labels, respectively. Furthermore gene expression patterns after 48 h were measured in mice. To elucidate the effect of different doses, all experiments were performed using ca. 1.5 microg and 25 microg of pDNA per mouse. Our studies demonstrated that both PEI and PEG-PEI form stable polyplexes with DNA with similar sizes of 100-130 nm. The zeta potential of PEI/pDNA polyplexes was highly positive, whereas PEG-PEI/pDNA showed a neutral surface charge as expected. The pharmacokinetic and organ distribution profiles after 2 h show similarities for both PEI and pDNA blood-level time curves from polyplexes at both doses indicative for significant stability in the bloodstream. A very rapid clearance from the bloodstream was observed and as major organs of accumulation liver and spleen were identified. PEG-PEI/pDNA complexes at a dose of approximately 25 microg exhibit similar profiles except a significantly lower deposition in the lung. At the lower dose of approximately 1.5 microg pDNA, however, for polyplexes from PEG-PEI, significant differences in blood level curves and organ accumulation of polymer and pDNA were found. In this case PEG-PEI shows a greatly enhanced circulation time in the bloodstream. By contrast, pDNA was rapidly cleared from circulation and significant amounts of radioactivity were found in the urine, suggesting a rapid degradation possibly by serum nucleases after complex separation. Regarding in vivo gene expression, no luciferase expression could be detected at approximately 1.5 microg dose in any organ using both types of complexes. At 25 microg only in the case of PEI/pDNA complexes were significant levels of the reporter gene detected in lung, liver, and spleen. This coincided with high initial accumulation of pDNA complexed with PEI and a high acute in vivo toxicity. For PEG-PEI, initial accumulation was much lower and no gene expression as well as a low acute toxicity was found. In summary, our data demonstrate that PEG-PEI used in this study is not suitable for low dose gene delivery. At a higher dose of approximately 25 microg, however, polyplex stability is similar to PEI/pDNA combined with a more favorable organ deposition and significantly lower acute in vivo toxicity. These findings have consequences for the design of PEG-PEI-based gene delivery systems for in vivo application.

The sympathetic control of blood supply is different in the spleen and lymph nodes.

OBJECTIVES: The noradrenergic innervation of lymphoid organs controls several immune cell functions and local blood perfusion. Considering that cell and antigen uptake depend on the blood supply to lymphoid organs, the hypothesis was tested that feedback signals from activated immune cells control sympathetic vasomotor activity. METHODS: We determined the blood flow in spleen and mesenteric lymph nodes (mLN) of Wistar Kyoto rats during immune stimulation with endotoxin (LPS; 10 microg/kg) and following disruption of the noradrenergic transmission. RESULTS: Our data indicate that (a) the splenic noradrenaline content, which reflects the density of the sympathetic innervation, is 5 times higher in the spleen than in other peripheral organs and the spleen receives stronger tonic sympathetic input than mLN; (b) immune stimulation with LPS causes a 4-fold increase in the IL-1beta production in the spleen, but only 2-fold in mLN; (c) IL-1beta causes an inhibition of the sympathetic vasoconstrictor tonus in the spleen, but has no significant effect on the noradrenergic vascular tonus in mLN, and (d) in mLN, the local hyperemia induced by LPS is attenuated by the degranulation of vesicular stores of histamine and serotonin, indicating that these monoamines participate in the vasodilator effect of LPS in mLN. CONCLUSIONS: The present experiments, taken together with our previous studies, indicate that the control of blood supply to the spleen and mLN involves different mechanisms. While blood perfusion in the spleen depends on the inhibition of the noradrenergic vasoconstriction by endogenously produced IL-1beta, other vasoactive mediators such as serotonin and histamine play a role in the control of mLN perfusion.

Local and systemic autonomic nervous effects on cell migration to the spleen.

This work is based on the hypothesis that sympathetic nerves regulate the uptake of circulating cells by the spleen by affecting splenic blood flow and that the quantity of cells sequestered depends on whether changes in noradrenergic transmission occur at local or systemic levels. Fluorescently labeled lymphoid cells were injected into rats, and organ blood flow was measured by the microsphere method. Increased retention of cells in the spleen paralleled by increased blood flow was detected after local denervation of this organ or administration of bacterial endotoxin. A comparable enhanced splenic blood flow was observed after general sympathectomy. However, the redistribution of blood perfusion during general vasodilatation resulted in deviation of leukocyte flow from the spleen, thus resulting in reduced uptake of cells by this organ. These results indicate that, although the uptake of cells by the spleen depends on arterial blood supply, enhanced perfusion does not always result in increased cell sequestration because general vasodilatation reduces cell uptake by this organ and even overrides stimulatory effects of endotoxin.