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The continually increasing wealth of knowledge about the role of genes involved in acquired or hereditary diseases renders the delivery of regulatory genes or nucleic acids into affected cells a potentially promising strategy. Apart from viral vectors, non‐viral gene delivery systems have recently received increasing interest, due to safety concerns associated with insertional mutagenesis of retro‐viral vectors. Especially cationic polymers may be particularly attractive for the delivery of nucleic acids, since they allow a vast synthetic modification of their structure enabling the investigation of structure‐function relationships. Successful clinical application of synthetic polycations for gene delivery will depend primarily on three factors, namely (1) an enhancement of the transfection efficiency, (2) a reduction in toxicity and (3) an ability of the vectors to overcome numerous biological barriers after systemic or local administration. Among the polycations presently used for gene delivery, poly(ethylene imine), PEI, takes a prominent position, due to its potential for endosomal escape. PEI as well as derivatives of PEI currently under investigation for DNA and RNA delivery will be discussed. This review focuses on structure‐function relationships and the physicochemical aspects of polyplexes which influence basic characteristics, such as complex formation, stability or in vitro cytotoxicity, to provide a basis for their application under in vivo conditions. Rational design of optimized polycations is an objective for further research and may provide the basis for a successful cationic polymer‐based gene delivery system in the future. Copyright © 2005 John Wiley & Sons, Ltd.

Time-resolved quantitative colocalization analysis is a method based on confocal fluorescence microscopy allowing for a sophisticated characterization of nanomaterials with respect to their intracellular trafficking. This technique was applied to relate the internalization patterns of nanoparticles i . e . superparamagnetic iron oxide nanoparticles with distinct physicochemical characteristics with their uptake mechanism, rate and intracellular fate. The physicochemical characterization of the nanoparticles showed particles of approximately the same size and shape as well as similar magnetic properties, only differing in charge due to different surface coatings. Incubation of the cells with both nanoparticles resulted in strong differences in the internalization rate and in the intracellular localization depending on the charge. Quantitative and qualitative analysis of nanoparticles-organelle colocalization experiments revealed that positively charged particles were found to enter the cells faster using different endocytotic pathways than their negative counterparts. Nevertheless, both nanoparticles species were finally enriched inside lysosomal structures and their efficiency in agarose phantom relaxometry experiments was very similar. This quantitative analysis demonstrates that charge is a key factor influencing the nanoparticle-cell interactions, specially their intracellular accumulation. Despite differences in their physicochemical properties and intracellular distribution, the efficiencies of both nanoparticles as MRI agents were not significantly different.

The interaction of nanoparticles (NP), consisting of hydrophobic polystyrene, bioadhesive chitosan, and stealth PLA-PEG with two human intestinal cell lines, the enterocyte-like Caco-2 and mucus-secreting MTX-E12, was investigated and compared to the in vivo NP uptake in rats. The extent and mechanism of cellular association of different NP with Caco-2 and MTX-E12 was investigated using confocal laser scanning microscopy (CLSM) and a cellular association assay. In vitro results were compared to gastrointestinal distribution of chitosan NP in rats after intra-duodenal injection. Cellular association of NP with Caco-2 cell monolayers showed the following rank order: polystyrene > chitosan >> PLA-PEG. Mucus (MTX-E12) significantly decreased the association of hydrophobic polystyrene NP. While no mucus binding was observed for PLA-PEG, association of chitosan NP with mucus strongly increased. Intra-duodenal administration of chitosan NP in rats confirmed these in vitro results, demonstrating that NP could be detected in both epithelial cells and Peyer's patches. Chitosan NP internalization was saturable, as well as energy- and temperature-dependent. It could be inhibited by an excess of protamine and by removal of anionic sites of the apical membrane. By contrast, polystyrene NP uptake was found to be largely independent of these factors, except for a temperature-dependency. In contrast to Caco-2 cells, the presence of mucus presented a major barrier for the uptake of hydrophobic polystyrene NP and showed an even more profound effect upon the uptake of chitosan NP. A correlation between the uptake in cell culture models and in vivo rat epithelial cells was confirmed for chitosan NP. Moreover, chitosan NP seemed to be taken up and transported by adsorptive transcytosis, while polystyrene NP uptake was probably mediated by non-adsorptive transcytosis.

Low molecular weight branched polyethylenimine (LMW-PEI) was synthesized and studied as a DNA carrier for gene delivery with regard to physico-chemical properties, cytotoxicity, and transfection efficiency. The architecture of LMW-PEI, synthesized by acid catalyzed ring-opening polymerization of aziridine was characterized by size exclusion chromatography in combination with laser light scattering and 13C-NMR-spectroscopy. In vitro cytotoxic effects were quantified by LDH and MTT assay and visualized by transmission electron microscopy. The potential for transgene expression was monitored in ECV304 cells using luciferase driven by a SV40 promotor as reporter gene system. LMW-PEI (Mw 11'900 D) with a low degree of branching was synthesized as a DNA carrier for gene delivery. In contrast to high molecular weight polyethylenimines (HMW-PEI; Mw 1'616'000 D), the polymer described here showed a different degree of branching and was less cytotoxic in a broad range of concentrations. As demonstrated by transmission electron microscopy the LMW-PEI formed only small aggregates which were efficiently taken up by different cells in the presence of serum, most likely by an endocytic pathway. LMW-PEI yielded transfection efficiencies measured via expression of the reporter gene luciferase which were up to two orders of magnitude higher than those obtained with HMW-PEI. The reporter gene expression was concentration dependent, but in contrast to lipofection independent of serum addition. The LMW-PEI described here is a new, highly efficient, and non-cytotoxic vector with a favorable efficiency/toxicity profile for gene therapeutic applications.

Background Targeting to integrin receptor ανβ3 by RGD peptides seems to be a promising approach for gene delivery to proliferating endothelial cells of tumor metastases. PEGylation of cationic polymers offers a reduction of non‐specific binding to cell surfaces. However, little knowledge exists on the influence of charge shielding by PEGylation on targeted gene delivery. Therefore, a variety of RGD peptide‐polyethylenimine (PEI) conjugates with different degrees of substitution, with or without poly(ethylene glycol) (PEG) spacer, were synthesized. Influence of degree of substitution and PEG spacer on physicochemical properties as well as on integrin targeting of DNA/polymer complexes was evaluated. Methods The tetrapeptide RGDC was coupled to PEI with or without a PEG spacer. Complex formation with DNA was monitored by ethidium bromide (EtBr) fluorescence quenching. Hydrodynamic diameters of complexes and zeta‐potential were assessed using a Zetasizer. Fluorescence correlation spectroscopy (FCS) was used to determine peptide binding to living cells. Transfection efficiency was evaluated employing a luciferase reporter gene. Binding of complexes to Mewo cells was monitored by flow cytometry. Results Polyplexes of RGD‐PEI or RGD‐PEG‐PEI and DNA showed reduced quenching of EtBr fluorescence compared with PEI. All RGD conjugates formed small polyplexes (approximately 100 nm in diameter at a nitrogen/phosphate (N/P) ratio of 6.7). At N/P = 6.7, the zeta‐potentials of RGD‐PEI complexes were similar to PEI complexes (25–30 mV), while RGD‐PEG‐PEI formed neutral complexes. FCS showed saturable binding of RGD peptide to Mewo human melanoma cells and only low binding to A549 human lung carcinoma cells. A degree of substitution of 4.6% with SPDP as coupling reagent yielded a conjugate showing 50 times higher luciferase expression in Mewo cells than unmodified PEI at low N/P ratios around 3.3, while a degree of substitution of 1.6% only led to a moderately increased transfection efficiency. Flow cytometry experiments suggest that this effect is partly caused by increased attachment of complexes to cell surfaces. No improvement in transfection efficiency was found in ανβ3‐negative A549 cells. RGD‐PEG‐PEI complexes showed reasonable transfection efficiencies at high N/P ratios; however, no targeting effect could be found. Conclusions Coupling of the tetrapeptide RGDC without a PEG spacer improved transfection efficiency of PEI in integrin‐expressing Mewo cells by 1–2 orders of magnitude, especially at low N/P ratios. The use of a PEG spacer seems to impair targeting, possibly by not only shielding PEI, but also the RGD ligand. Copyright © 2003 John Wiley & Sons, Ltd.

Present studies are carried out with an aim to make degradable materials based on caprolactone and vinyl acetate units using radical chemistry. Radical ring-opening copolymerization of 2-methylene-l,3-dioxepane (MDO) with vinyl acetate in presence of AIBN initiator at 70 °C was carried out to achieve the aim. The copolymerization introduced degradable PCL repeat units onto the C-C backbone of poly(vinyl acetate). Microstructure analysis of the copolymers is done using different ID and 2D NMR techniques. Complete ring-opening polymerization of MDO to give ester units was observed during copolymerizations. Reactivity ratios were found out by Kelen Tüdos method and were r VAc = 1.53 and r MDO = 0.47 leading to statistical introduction of ester linkages onto the polymer backbone. The materials showed varied glass transition temperatures (from 37 to −44°C) depending upon the amount of ester linkages and very high elongations. The hydrolysis products were also tested for cytotoxicity studies in L929 cells and compared with that of known and accepted standard materials like poly(ethyleneimine). The hydrolysed products were non toxic and showed a cell viability > 95%. Keeping in view the combined properties like degradability, non-toxicity and low glass transition temperatures, the resulting materials could therefore be proposed for different applications like degradable gums, coatings etc.

Purpose The objective of this study was to investigate how the degree of amine substitution of amine-modified poly(vinyl alcohol) (PVA) affects complexation of siRNA, protection of siRNA against degrading enzymes, intracellular uptake and gene silencing. Methods A series of DEAPA-PVA polymers with increasing amine density was synthesized by modifying the hydroxyl groups in the PVA backbone with diethylamino propylamine groups using CDI chemistry. These polymers were characterized with regard to their ability to complex and protect siRNA against RNase. Finally, their potential to mediate intracellular uptake and gene silencing in SKOV-luc cells was investigated. Results A good correlation between amine density and siRNA complexation as well as protection of siRNA against RNase was found. Consisting solely of tertiary amines, this class of polymer was able to mediate efficient gene silencing when approximately 30% of the hydroxyl groups in the PVA backbone were modified with diethylamino propylamine groups. Polymers with a lower amine density (up to 23%) were inefficient in gene silencing, while increasing the amine density to 48% led to non-specific knockdown effects. Conclusion DEAPA-PVA polymers were shown to mediate efficient gene silencing and offer a promising platform for further structural modifications.

Abstract Mucociliary clearance (MC), designed by evolution to eliminate inhaled and possibly noxious material from the airways, considerably limits the benefit of inhalation therapy. Although the principles of MC seem to be understood, there are still many open questions on mucociliary particle clearance. In this study a trachea-based in vitro model was used to investigate the effect of particle size, zeta-potential, and mucoadhesive particle properties on mucociliary particle clearance. As different sized particles (50–6000 nm) were tested at equal mass concentrations, size related factors, namely particle number and particle surface area, varied by several orders of magnitude between the experiments. Surprisingly, particle clearance for 50 nm up to 6000 nm-sized polystyrene particles did not differ significantly (p < 0.05): 50 nm (2.9 ± 0.6 mm/min); 100 nm (3.8 ± 0.9 mm/min); 1000 nm (3.8 ± 0.8 mm/min); 6000 nm (3.2 ± 0.6 mm/min). In clear contrast, particles prepared from different PLGA-based copolymers (polylactic-co-glycolic acid) showed a significant effect on particle transport. PEG-PLGA particles (polyethylene glycol) showed the fastest and normal transport rates (5.9 ± 1.7 mm/min) compared to the ICRP's (International Commission of Radiological Protection) standard value for average tracheal transport rates (5.5 mm/min). Mucoadhesive chitosan-PLGA particles were transported at the slowest rate (0.7 ± 0.3 mm/min) of all particles tested. Overall, particle size and zeta-potential seem to be relatively uncritical, whereas material properties and the related particle surface chemistry significantly influence mucociliary particle clearance. Considering these findings in future drug formulation seems to be a promising strategy to improve inhalation therapy by prolonged particle/drug residence time within the airways.

A new mucus-secreting in vitro drug absorption model based on monolayers of goblet-cell like sub-clones of the human colon carcinoma cell line HT29 obtained by methotrexate (MTX) treatment was investigated. Twelve sub-clones were isolated and characterized by light microscopy (LM), transelectron microscopy (TEM), confocal laser scanning microscopy (CLSM), transepithelial electrical resistance (TEER) and the transport of a paracellular marker FITC-Dextran (Mw 4400) (FD-4). Significant differences of microscopical appearance, TEER-values and permeability of FD-4 between the sub-clones were evident. However, two of them, namely MTX-D1 and MTX-E12. formed tight confluent monolayers with a thick mucus-layer on the apical surface. They were used to compare the apparent permeability coefficient (Papp) of a series of lipophilic drugs, which should be affected by the mucus-layer, namely barbiturates (barbituric acid, barbital, phenobarbital, methylphenobarbital and heptabarbital) and testosterone, as a reference, to mucus-free Caco-2 cells. The permeability of drugs with a partition coefficient (log P) > 1 was decreased in the mucus-producing cell lines. Testosterone, the most lipophilic compound, showed a decrease of up to 43%. We demonstrated that the mucus layer is a significant barrier to drug absorption for lipophilic drugs. In conclusion, our model may serve as a suitable in-vitro cell culture model to study the influence of the mucus layer on drug diffusion.

To investigate the effect of locally applied nimodipine prolonged-release microparticles on angiographic vasospasm and secondary brain injury after experimental subarachnoid hemorrhage (SAH). 70 male Wistar rats were categorized into three groups: 1) sham operated animals (control), 2) animals with SAH only (control) and the 3) treatment group. SAH was induced using the double hemorrhage model. The treatment group received different concentrations (20%, 30% or 40%) of nimodipine microparticles. Angiographic vasospasm was assessed 5 days later using digital subtraction angiography (DSA). Histological analysis of frozen sections was performed using H&E-staining as well as Iba1 and MAP2 immunohistochemistry. DSA images were sufficient for assessment in 42 animals. Severe angiographic vasospasm was present in group 2 (SAH only), as compared to the sham operated group (p<0.001). Only animals within group 3 and the highest nimodipine microparticles concentration (40%) as well as group 1 (sham) demonstrated the largest intracranial artery diameters. Variation in vessel calibers, however, did not result in differences in Iba-1 or MAP2 expression, i.e. in histological findings for secondary brain injury. Local delivery of high-dose nimodipine prolonged-release microparticles at high concentration resulted in significant reduction in angiographic vasospasm after experimental SAH and with no histological signs for matrix toxicity.