Epidermal growth factor-induced modulation of cytokeratin expression levels influences the morphological phenotype of head and neck squamous cell carcinoma cells.

The migratory ability of tumor cells requires cytoskeletal rearrangement processes. Epidermal growth factor receptor (EGFR)-signaling tightly correlates with tumor progression in head and neck squamous cell carcinomas (HNSCCs), and has previously been implicated in the regulation of cytokeratin (CK) expression. In this study, HNSCC cell lines were treated with EGF, and CK expression levels were monitored by Western blot analysis. Changes in cellular morphology were documented by fluorescence- and atomic force microscopy. Some of the cell lines demonstrated an EGF-dependent modulation of CK expression levels. Interestingly, regression of some CK subtypes or initial up-regulation followed by downregulation at higher EGF-levels could also be observed in the tested cell lines. Overall, the influence of EGF on CK expression levels appeared variable and cell-type-dependent. Real-time cellular analysis of EGF-treated and -untreated HNSCC cell lines demonstrated a rise over time in cellular impedance. In three of the EGF-treated HNSCC cell lines, this rise was markedly higher than in untreated controls, whereas in one of the cell lines the gain of cellular impedance was paradoxically reduced after EGF treatment, which was found to correlate with changes in cellular morphology rather than with relevant changes in cellular viability or proliferation. After treating HNSCC cells with EGF, CK filaments frequently appeared diffusely distributed throughout the cytoplasm, and in some cases were found in a perinuclear localization, the latter being reminiscent to observations by other groups. In summary, the data points to a possible role of EGFR in modulating HNSCC cell morphology.

Predicting protein instability in sustained protein delivery systems using spectral-phase interference.

Biodegradable and non-biodegradable polymers represent promising materials for sustained protein delivery systems. However, structural protein instabilities due to interactions with the polymer surface are often observed. Aim of the present study was to analyze and predict these instabilities by determination of adsorption pattern and extent via biomolecular interaction analysis. A new optical method based on spectral-phase interference successfully demonstrated its suitability for this new application scope. It was characterized in terms of sensitivity, reproducibility and dynamic range using bovine serum albumin (BSA) as model compound. For protein-polymer interaction studies, materials with different wettabilities and zeta potential were selected and successfully applied on the sensor chip: Glass, poly(styrene), poly(lactic acid), poly(lactic-co-glycolic acid), and poly(ethylene carbonate). Concentration dependent adsorption curves revealed two principal adsorption patterns based on the connection between BSA spreading and supply rate. This connection was stronger influenced by polymer hydrophobicity than surface charge. Association, dissociation and binding rate constants in the range from 0.15 to 34.19 × 10(-6) M were obtained. Atomic force microscopy images of the films before and after adsorption confirmed the previous elaborated model. Poly(ethylene carbonate) emerged as highly promising biomaterial for protein delivery due to its favorable adsorption behavior based on low polymer-protein interactions.

Methotrexate-loaded chitosan- and glycol chitosan-based nanoparticles: a promising strategy for the administration of the anticancer drug to brain tumors.

Brain tumor treatment employing methotrexate (MTX) is limited by the efflux mechanism of Pg-p on the blood-brain barrier. We aimed to investigate MTX-loaded chitosan or glycol chitosan (GCS) nanoparticles (NPs) in the presence and in the absence of a coating layer of Tween 80 for brain delivery of MTX. The effect of a low Tween 80 concentration was evaluated. MTX NPs were formulated following the ionic gelation technique and size and zeta potential measurements were acquired. Transport across MDCKII-MDR1 monolayer and cytotoxicity studies against C6 glioma cell line were also performed. Cell/particles interaction was visualized by confocal microscopy. The particles were shown to be cytotoxic against C6 cells line and able to overcome MDCKII-MDR1 cell barrier. GCS-based NPs were the most cytotoxic NPs. Confocal observations highlighted the internalization of Tween 80-coated fluorescent NPs more than Tween 80-uncoated NPs. The results suggest that even a low concentration of Tween 80 is sufficient for enhancing the transport of MTX from the NPs across MDCKII-MDR1 cells. The nanocarriers represent a promising strategy for the administration of MTX to brain tumors which merits further investigations under in vivo conditions.

Stability of monomolecular films of archaebacterial tetraether lipids on silicon wafers: a comparison of physisorbed and chemisorbed monolayers.

The monomolecular organisation of symmetric, chemically modified tetraether lipids caldarchaeol-PO(4) was studied using Langmuir film balance, ellipsometry, and atomic force microscopy (AFM). Solid silicon wafer substrates were modified to hydrophobic, hydrophilic, and amino-silanised surfaces; and Langmuir-Blodgett (LB)-films were transferred onto each. LB-caldarchaeol-PO(4) films were subjected to further rinsing with organic solvent and additional physical treatments, to compare their resistance and stability on chemisorbed (amino-silanised) and physisorbed (hydrophobic and hydrophilic) surfaces. The resistance and stability of these monolayer films was characterized by ellipsometry and AFM, and film thickness was determined using ellipsometry. AFM was also employed to observe surface morphology. Monolayer films on hydrophobic surfaces were found to be more resistant to rinsing with organic solvent and additional physical treatments than monolayer films on either amino-silanised or hydrophilic surfaces. The hydrophobic effect with hydrophobic surfaces appears to support the formation of stronger caldarchaeol-PO(4) films on silicon wafer substrates, with increased resistance and stability.

AFM and ellipsometric studies on LB films of natural asymmetric and symmetric bolaamphiphilic archaebacterial tetraether lipids on silicon wafers.

The monomolecular organisation of the natural asymmetric and symmetric bolaamphiphilic (archaebacterial tetraether lipids) was studied using a combination of Langmuir film balance, ellipsometry, and atomic force microscopy (AFM). Monolayer films were transferred onto silicon wafer substrates. After transfer of the lipids onto several kinds of silicon wafer substrates (hydrophobic, hydrophilic, and amino-silanised), the monolayer films were further investigated by ellipsometry and AFM in order to determined whether bolaamphiphiles are oriented differently at the water-air interface, including "horseshoe" and "upright" configurations. The thickness of the monolayer film, which was determined by ellipsometry, represents a combined mean value of the domains. The surface morphology, which was evaluated by AFM showed that in all films, the domains were arranged in small domains or the most homogeneous organisation, depending on the properties of the wafer silicon substrates. From all films, the hydrophobically transferred lipids showed the most homogeneous organisation on the substrates. After transfer onto hydrophilic and amino-silanised surfaces, the lipids were arranged in small islands on the substrates.

Utilising atomic force microscopy for the characterisation of nanoscale drug delivery systems.

The introduction of atomic force microscopy (AFM) techniques has revolutionised our ability to characterise colloidal objects. AFM allows the visualisation of samples with sub-nanometre resolution in three dimensions in atmospheric or submerged conditions. Nanomedical research is increasingly focused on the design, characterisation and delivery of nano-sized drug carriers such as nanoparticles, liposomes and polyplexes, and this review aims to highlight the scope and advantages of AFM in this area. A significant amount of work has been carried out in drug delivery system (DDS) research in recent years using a large variety of techniques. The use of AFM has enabled us to directly observe very small objects without the need of a cumbersome and potentially contaminating sample preparation. Thus, nanoscale DDS can be investigated in a controlled environment without the necessity of staining or drying. Moreover, intermittent contact mode AFM allows the investigation of soft samples with minimal sample alteration; phase imaging allows accessing information beyond the sample's topography and also differentiating between different materials, and force spectroscopy experiments help us to understand the intrinsic structure of DDS by recording the elastic or adhesion behaviour of particles. Hence, AFM enables us accessing information which is hardly available by other experimental techniques. It has provided invaluable information about physicochemical properties and helped to shed light on the area of nanoscale drug delivery and will, with more and more sophisticated equipment becoming available, continue to add to our understanding of the behaviour of nanoscale DDS in the future.

Atomic force microscopy for the characterization of proteoliposomes.

Atomic Force Microscopy (AFM) is a useful tool for the visualization of soft biological samples in a nanoscale resolution. In the study presented here, the surface morphology ofP-selectin and Transferrin modified proteoliposomes were investigated in air and under water. The proteins were visualized without pre-functionalization or staining.

Triazine dendrimers as nonviral gene delivery systems: effects of molecular structure on biological activity.

A family of generation 1, 2, and 3 triazine dendrimers differing in their core flexibility was prepared and evaluated for their ability to accomplish gene transfection. Dendrimers and dendriplexes were analyzed by their physicochemical and biological properties such as condensation of DNA, size, surface charge, morphology of dendriplexes, toxic and hemolytic effects, and ultimately transfection efficiency in L929 and MeWo cells. Flexibility of the backbone was found to play an important role with generation 2 dendrimer displaying higher transfection efficiencies than 25 kDa poly(ethylene imine) or SuperFect at a lower cytotoxicity level. This result is surprising, as PAMAM dendrimers require generations 4 or 5 to become effective transfection reagents. The ability to delineate effects of molecular structure and generation of triazine dendrimers with biological properties provides valuable clues for further modifying this promising class of nonviral delivery system.

The potential of glycol chitosan nanoparticles as carrier for low water soluble drugs.

The laser dye 6-coumarin was selected as model of low water soluble drug to be encapsulated in glycol chitosan nanoparticles intended for transmucosal applications and, at the same time, being a fluorescent probe, it is of aid to elucidate the intracellular fate of the particles. To increase the aqueous solubility of the tracer, the complexation with different cyclodextrins was adopted. The fluorescence properties of the inclusion complexes were evaluated. The increase in aqueous solubility provided by different cyclodextrins [up to 1.4x10(-4) M in the case of heptakis (2,6-di-O-methyl)-beta-cyclodextrin] allowed the preparation of novel glycol chitosan nanoparticles according to the ionic cross-linking of the polycation by sodium tripolyphosphate. Small changes in the preparation technique allowed to produce particles of two different sizes, around 200 nm and bigger than 300 nm where the contribution of cyclodextrin consisted of the modulation encapsulation efficiency in the final particles. Confocal laser scanning microphotographs clearly showed the internalization of 6-coumarin nanoparticles in Caco-2 cell line. The results reveal that these biodegradable nanoparticles hold promise as probes in biomedical field.

Stabilization of aerosolizable nano-carriers by freeze-drying.

PURPOSE: This study investigates the feasibility of freeze-drying aerosolizable nano-carriers (NC) by the use of different lyoprotective agents (LPA) and the influence of the freeze-drying on the physicochemical properties of these nano-carriers and on their aerosolization. METHODS: Nano-carriers were prepared from fast-degrading polymers, DMAPA(24)-PVAL-g-PLGA(1:7.5) and DEAPA(26)-PVAL-g-PLGA(1:10), and freeze-dried using increasing concentrations of different LPA. The hydrodynamic diameter, zeta potential and morphology (atomic force microscopy) of NC were characterized before and after freeze-drying. The ability to aerosolize using a jet nebulizer and an electronic micro-pump nebulizer was also investigated. RESULTS: Freeze-drying with LPA led to a decreased zeta-potential of NC and changes in size about 20 nm without alteration in shape, whereas lyophilizates without LPA were found to aggregate. While freeze-drying was positively affected by increasing concentrations, it was not influenced by the type of LPA. The possibility for aerosolization was not influenced by any LPA. CONCLUSIONS: Freeze-drying with LPA is a suitable method to physically stabilize fast-degrading NC from aqueous suspensions without influencing the aerosolizability.

Effects of cell-penetrating peptides and pegylation on transfection efficiency of polyethylenimine in mouse lungs.

BACKGROUND: Cell-penetrating peptides (CPPs) could potentially be used as vectors for intracellular delivery of proteins, peptides and nucleic acids. The present study examined different CPPs, such as TAT-derived and arginine rich sequences, as well as model amphiphilic peptide, with respect to transfection efficiency of pegylated polyethylenimine (PEI) in A549, Calu-3 cells and in mice after intra-tracheal administration. METHODS: The conjugates were prepared by the coupling of CPPs to PEI via a heterobifunctional polyethylene glycol (PEG) linker, resulting in the bioconjugates CPP-PEG-PEI. Structures were successfully confirmed by (1)H-nuclear magnetic resonance and diffusion-ordered spectroscopy. Unmodified PEI 25 kDa was compared with pegylated PEI, and aggregation tendency in cell culture medium, interaction with mucin and stability against heparin was assayed. After evaluating transfection efficiency of the polymers in two different lung cell lines, luciferase reporter gene expression was determined in mouse lungs. RESULTS: All conjugates showed superior transfection efficiency compared to unmodified PEI 25 kDa. The conjugates sizes were generally < 300 nm, thus enabling them to penetrate through the mucus lining of the lung and reach the target cells. Coupling of CPPs to PEG-PEI, however, did not significantly improve transfection efficiency in A549 cells, calu-3 cells and in mouse lungs. CONCLUSIONS: We show that small and stable polyplex size achieved by pegylation is favourable for successful pulmonary gene delivery. Compared to PEI 25 kDa, pegylated PEI and CPP-PEG-PEI displayed enhanced transfection efficiency both in vitro and in vivo.

Polyethylenimine PEI F25-LMW allows the long-term storage of frozen complexes as fully active reagents in siRNA-mediated gene targeting and DNA delivery.

BACKGROUND: Polyethylenimines (PEIs) are synthetic, charged polymers which function as transfection reagents based on their ability to compact DNA into complexes. Recently, PEI-mediated delivery of nucleic acids has been extended towards small interfering RNAs (siRNAs) which are instrumental in the induction of RNA interference (RNAi). Since RNAi represents a powerful method for specific gene silencing, the PEI-based delivery of siRNAs is a promising tool for novel putative therapeutic strategies. AIM: For therapeutic use, major requirements are the development of formulations which (i) are sufficiently stable in the presence of serum, and which can be (ii) easily and reproducibly manufactured and (iii) stored for a prolonged time with full retention of their integrity and bioactivity. In this paper, we explore the potential of PEI F25-LMW, a low-molecular weight PEI with superior transfection efficacy and low toxicity, towards these goals. RESULTS: We have systematically analyzed and determined optimal DNA and siRNA complexation conditions with regard to various parameters including buffer concentration, ionic strength, pH and incubation time. As opposed to 22kDa linear PEI (L-PEI), the low-molecular weight (4-10kDa) PEI F25-LMW performs DNA transfection and siRNA gene targeting with identical efficacies in the presence of serum, thus emphasizing its usefulness in vivo. Furthermore, in contrast to other polyethylenimines, PEI F25-LMW-based DNA or siRNA complexes allow freeze/thawing and frozen storage for several months. Their activity is fully retained without requiring specific buffer conditions or the addition of any lyoprotectant. Physicochemical analysis and atomic force microscopy reveal a distinct size pattern with the presence of two complex subgroups and show that frozen PEI F25-LMW complexes remain stable with little increase in complex size, no changes regarding their zeta potential and cytotoxicity, and full retention of nucleic acid protection. CONCLUSIONS: Frozen PEI F25-LMW-based complexes represent efficient and stable ready-to-use formulations of DNA- or siRNA-based gene therapy products.

Gene delivery using chitosan, trimethyl chitosan or polyethylenglycol-graft-trimethyl chitosan block copolymers: establishment of structure-activity relationships in vitro.

Chitosan, trimethyl chitosan or polyethylenglycol-graft-trimethyl chitosan/DNA complexes were characterized concerning physicochemical properties such as hydrodynamic diameter, condensation efficiency and DNA release. Furthermore, cytotoxicity of polymers and uptake- and transfection efficiency of polyplexes were evaluated in vitro. Under conditions found in cell culture, formation of aggregates of approximately 1000 nm and strongly decreased DNA condensation efficiency was observed in the case of chitosan polyplexes. These characteristics resulted in only 7% cellular uptake in NIH/3T3 cells and low transfection efficiencies in 4 different cell lines. By contrast, quaternization of chitosan strongly reduced aggregation tendency and pH dependency of DNA complexation. Accordingly, cellular uptake was increased 8.5-fold compared to chitosan polyplexes resulting in up to 678-fold increased transfection efficiency in NIH/3T3 cells. Apart from reduction of the cytotoxicity, PEGylation led to improved colloidal stability of polyplexes and significantly increased cellular uptake compared to unmodified trimethyl chitosan. These improvements resulted in a significant, up to 10-fold increase of transfection efficiency in NIH/3T3, L929 and MeWo cells compared to trimethyl chitosan. This study not only highlights the importance of investigating polyplex stability under different pH- and ionic strength conditions but also elucidates correlations between physicochemical characteristics and biological efficacy of the studied polyplexes.

Peroral delivery of insulin using chitosan derivatives: a comparative study of polyelectrolyte nanocomplexes and nanoparticles.

Polymeric delivery systems based on nanoparticles (NP) have emerged as a promising approach for peroral insulin delivery. Using a trimethyl chitosan (TMC) and a PEG-graft-TMC copolymer, polyelectrolyte complexes (PEC) and nanoparticles were prepared and their properties were compared. The amount of insulin was quantified by HPLC and the stability of PEC and NP upon exposure to simulated gastrointestinal (GI) fluid was monitored by dynamic laser light scattering. It was shown that polymer/insulin (+/-) charge ratio played an important role in PEC and NP formation. Stable, uniform, and spherical PEC/NP with high insulin association efficiency (AE) were formed at or close to optimized polymer/insulin (+/-) charge ratio, depending on polymer structure. All PEC were more stable in pH 6.8 simulated intestinal fluid (SIF) than NP. The PEC also appeared to play some role in protecting insulin from degradation at higher temperature and with proteolytic enzyme more efficiently than NP. On the basis of these results, polyelectrolyte complexation can be suggested as a potentially useful technique for generating insulin delivery systems for peroral administration.

Stabilized nanocarriers for plasmids based upon cross-linked poly(ethylene imine).

Stabilized PEI/DNA polyplexes were generated by cross-linking PEI with biodegradable disulfide bonds. The reaction conversion of different PEIs with the amine reactive cross-linker dithiobis(succinimidyl propionate) (DSP) was investigated, and the molecular weight of the reaction products was identified. Light scattering and microelectrophoresis were employed to assess size and zeta potential of the resulting polyplexes. Polyplex morphology and mechanic stability were investigated using atomic force microscopy. Finally, albumin and erythrocyte interactions and stability against polyanions and high ionic strength were checked. Polyplexes of PEI and DNA were prepared by two different formulation methods, either using pre-cross-linked polymers or by cross-linking polyplexes after complexation. Only the latter method yielded small (100-300 nm) polyplexes with a positive zeta potential when HMW PEI was used, whereas cross-linked LMW PEI resulted in polyplexes with increased size (>1000 nm) and zeta potentials down to -20 mV. In addition, only cross-linking after polyplex formation was able to enhance resistance against polyanion exchange and high ionic strength. AFM images revealed no changes in the morphology of cross-linked HWM PEI polyplexes, and indentation force measurements using AFM significantly increased mechanical stability of cross-linked HMW PEI polyplexes. These polyplexes also displayed significantly reduced interactions with major blood components like albumin and erythrocytes. The resulting biocompatible particles offer a means of combining enhanced polyplex stability with redox-triggered activation for in vivo application.

Influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft-poly(ethylene glycol) block copolymer/SiRNA polyplexes.

Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio ( approximately 50%) on the biophysical properties of the polyplexes. Particle size, zeta potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA polyplexes were characterized. Stability and size of siRNA polyplexes were clearly influenced by PEI-PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)(30) resulted in large (300-400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA polyplexes against heparin displacement and RNase digestion could be modified by PEGylation. Protection against RNase digestion was highest for PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1), while siRNA/PEI provided insufficient protection. In knockdown experiments using NIH/3T3 fibroblasts stably expressing beta-galactosidase, it was shown that PEG chain length had a significant influence on biological activity of siRNA. Polyplexes with siRNA containing PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1) yielded similar efficiencies of ca. 70% knockdown as lipofectamine controls. Confocal microscopy demonstrated enhanced cellular uptake of siRNA into cytosol by polyplexes formation with PEI copolymers. In conclusion, both the chain length and graft density of PEG were found to strongly influence siRNA condensation and stability and hence affect the knockdown efficiency of PEI-PEG/siRNA polyplexes.