Progressive external ophthalmoplegia (PEO) can be caused by a disorder characterized by multiple mitochondrial DNA (mtDNA) deletions due to mutations in the TWINKLE gene, encoding a mtDNA helicase. We describe a 71-year-old woman who had developed PEO at age 55 years. She had cataracts, diabetes, paresthesias, cognitive defects, memory problems, hearing loss, and sensory ataxia. She had muscle weakness with ragged red fibers on biopsy. MRI showed static white matter changes. A c.908G>A substitution (p.R303Q) in the TWINKLE gene was identified. Multiple mtDNA deletions were detected in muscle but not blood by a PCR-based method, but not by Southern blot analysis. MtDNA copy number was maintained in blood and muscle. A systematic literature search was used to identify the genotypic and phenotypic spectrum of dominant TWINKLE-related disease. Patients were adults with PEO and symptoms including myopathy, neuropathy, dysarthria or dysphagia, sensory ataxia, and parkinsonism. Diabetes, cataract, memory loss, hearing loss, and cardiac problems were infrequent. All reported mutations clustered between amino acids 303 and 508 with no mutations at the N-terminal half of the gene. The TWINKLE gene should be analyzed in adults with PEO even in the absence of mtDNA deletions in muscle on Southern blot analysis, and of a family history for PEO. The pathogenic mutations identified 5' beyond the linker region suggest a functional role for this part of the protein despite the absence of a primase function in humans. In our patient, the pathogenesis involved multiple mtDNA deletions without reduction in mtDNA copy number.

The poly(ethylene oxide)-poly(lactide) (PEO-PLA) block copolymers containing a small quantity of carboxylic acid in the PLA block were synthesized. The microscopic characteristics of nanoparticles with carboxylic acid content in the copolymer were analyzed, and the effect of specific interactions between the copolymer and the model drug on the drug loading capacity and the release behavior were investigated systematically. The sizes of nanoparticles prepared by a dialysis method are within the range of 30-40 nm. The nanoparticles prepared from functionalized block copolymers have a very low critical micelle concentration (CMC) value as low as approximately 10(-3) mg/ml, which indicates a good stability of the nanoparticles in spite of the presence of carboxylic acid. The drug loading efficiency of nanoparticles dramatically increased when carboxylic acid content was increased in the block copolymer. This result may be attributed to the increase of interactions between the copolymer and the drug. The release rate of the drug was much slower from nanoparticles containing higher amounts of carboxylic acid in the copolymer, which might be associated with the enhanced interaction between the carboxylic group of copolymers and the drug. These experimental results suggest that the nanoparticles prepared from functionalized PEO-PLA block copolymers could be a good candidate for an injectable drug delivery carrier.

This study evaluated differences in sitting habits in the classroom between the project "Moving school" and a traditional school in 8-year-old children. Twenty-two children, since 1.5 years involved in the project were compared to 25 children in a traditional school. Making use of the Portable Ergonomic Observation (PEO) method, it was observed that children from a traditional school spend an average of 97% of the lesson time sitting statically, from which one-third with the trunk bend over 45 degrees. In the "Moving school" this posture was replaced by dynamic sitting (53%), standing (31%) and walking around (10%), while trunk flexion over 45 degrees was nearly not observed. Children from the "Moving school" also showed significantly less neck and trunk rotation. Additionally, accelerometric data showed significantly more physical activity in lessons of the "Moving school". Rates of self-reported back or neck pain did not differ significantly between both study groups. Results show that sitting habits are more favourable in a "Moving school". Further research is needed to study the impact of implementing "Moving school" concepts in traditional schools on sitting habits.

Poly(ethylene oxide) (PEO) was used to prepare thin polymer films containing clotrimazole (CT) utilizing hot-melt extrusion (HME) technology. Films containing PEOs of two different molecular weights and the drug were investigated for solid-state characteristics, moisture-sorption, bioadhesivity, mechanical properties, release characteristics, and physical and chemical stability of the drug within the HME films. The solid-state characterization of the drug and the polymer were performed utilizing differential scanning calorimetry and X-ray diffractometry. A Texture analyzer was utilized to study the bioadhesive and mechanical properties of the HME films. Physical and chemical stability of the films, stored at 25 degrees C/60% RH, was studied for up to 12 months. XRD profiles indicated that the drug was physically unstable (recrystallization of the drug occurred) after storage for 3 months at 25 degrees C/60% RH. Based on the DSC studies, it has been proposed that the recrystallization of the drug may be due to the folding (due to HME) and unfolding (upon storage) of the linear PEO chains. Desirable bioadhesive, mechanical, and thermoplastic properties of PEO qualify it as a promising and potential drug carrier. However, further investigation is necessary to enhance the physical stability of these PEO-drug systems.

OBJECTIVE

To assess the intracellular delivery, antiretroviral activity and cytotoxicity of poly(ε-caprolactone) (PCL) nanoparticles containing the antiretroviral drug dapivirine.

METHODS

Dapivirine-loaded nanoparticles with different surface properties were produced using three surface modifiers: poloxamer 338 NF (PEO), sodium lauryl sulfate (SLS) and cetyl trimethylammonium bromide (CTAB). The ability of nanoparticles to promote intracellular drug delivery was assessed in different cell types relevant for vaginal HIV transmission/microbicide development. Also, antiretroviral activity of nanoparticles was determined in different cell models, as well as their cytotoxicity.

RESULTS

Dapivirine-loaded nanoparticles were readily taken up by different cells, with particular kinetics depending on the cell type and nanoparticles, resulting in enhanced intracellular drug delivery in phagocytic cells. Different nanoparticles showed similar or improved antiviral activity compared to free drug. There was a correlation between increased antiviral activity and increased intracellular drug delivery, particularly when cell models were submitted to a single initial short-course treatment. PEO-PCL and SLS-PCL nanoparticles consistently showed higher selectivity index values than free drug, contrasting with high cytotoxicity of CTAB-PCL.

CONCLUSIONS

These results provide evidence on the potential of PCL nanoparticles to affect in vitro toxicity and activity of dapivirine, depending on surface engineering. Thus, this formulation approach may be a promising strategy for the development of next generation microbicides.

The goal of this study was to assess a solvent evaporation method for the encapsulation of amphotericin B (AmB) in poly(ethylene oxide)-block-poly(N-hexyl stearate L-aspartamide) (PEO-b-PHSA) micelles. By the solvent evaporation method, PEO-b-PHSA self-assembled into small spherical micelles with a high AmB content based on transmission electron microscopy, size exclusion chromatography and absorption spectroscopy. The encapsulation of AmB was slightly better than an earlier method based on dialysis. Importantly, AmB in PEO-b-PHSA micelles encapsulated by the solvent evaporation method was non-haemolytic at 15 microg/ml, whereas AmB in PEO-b-PHSA micelles encapsulated by the dialysis method caused 50% haemolysis at the level of 3.8 microg/ml, and AmB itself caused 100% haemolysis at 1.0 microg/ml. Thus, PEO-b-PHSA micelles could effectively encapsulate AmB, increase the overall water solubility of AmB and reduce the toxicity of the membrane-acting drug, particularly by a solvent evaporation method.

Only four different mutations in the adenine nucleotide translocator 1 (ANT1) gene have been found in families with progressive external ophthalmoplegia (PEO). We report a novel heterozygous C to A transversion at nucleotide 269 in the ANT1 gene in a German family with PEO, predicted to convert a highly conserved alanine at codon 90 to aspartic acid. The mutation was identified in three siblings with PEO, one of them additionally suffered from schizoaffective disorder. Microsatellite analysis showed that the mutation was dominant and inherited from the mother who did not carry the mutation in blood, indicating germ-line mosaicism.

Nonwoven fiber mats of chitosan with potential applications in air and water filtration were successfully made by electrospinning of chitosan and poly(ethyleneoxide) (PEO) blend solutions. Electrospinning of pure chitosan was hindered by its limited solubility in aqueous acids and high degree of inter- and intrachain hydrogen bonding. Nanometer-sized fibers with fiber diameter as low as 80 +/- 35 nm without bead defects were made by electrospinning high molecular weight chitosan/PEO (95:5) blends. Fiber formation was characterized by fiber shape and size and was found to be strongly governed by the polymer molecular weight, blend ratios, polymer concentration, choice of solvent, and degree of deacetylation of chitosan. Weight fractions of polymers in the electrospun nonwoven fibers mats were determined by thermal gravimetric analysis and were similar to ratio of polymers in the blend solution. Surface properties of fiber mats were determined by measuring the binding efficiency of toxic heavy metal ions like chromium, and they were found to be related with fiber composition and structure.

Films of three ABA-block copolymers composed of lactic-co-glycolic acid A blocks and poly(oxyethylene) (PEO) B blocks and one random lactic-co-glycolic acid copolymer (PLG) were studied to investigate the influence of different polymer compositions and molecular weights on the tissue reaction, appearance of toxic degradation products, and swelling behavior in the cage implant system in rats. The inflammatory tissue reaction was followed over a 21-day implantation period by monitoring the leukocyte concentration, the extracellular acid, and alkaline phosphatase activities in a quantitative manner. Size and density of adherent macrophages and foreign body giant cells on the film surfaces were determined. The ABA and PLG implants caused only a minimal inflammatory reaction, as characterized by a low concentration of leukocytes during the implantation period when compared to empty cage controls. The content of PEO had an influence on the density of the adherent cells on the surface of the polymer film. An increase in PEO content and molecular weight decreased the cellular density during the implantation period. As demonstrated by scanning electron microscopy, no degradation was observed for all polymers during the implantation period. Our results demonstrate that the ABA block copolymers and PLG copolymer, are equally well tolerated in the cage implant test system.

Linear, biodegradable, aliphatic polyurethanes with various degrees of hydrophilicity were synthesized in bulk at 50-100 degrees C. The ratios between the hydrophilic and hydrophobic segments were 0:100, 30:70, 40:60, 50:50, and 70:30, respectively. The hydrophilic segment consisted of poly(ethylene oxide) (PEO) diol (molecular weight = 600 or 2000) or the poly(ethylene-propylene-ethylene oxide) (PEO-PPO-PEO) diol Pluronic F-68 (molecular weight = 8000). The hydrophobic segment was made of poly(epsilon-caprolactone) diol (molecular weight = 530, 1250, or 2000). The chain extenders were 1,4-butane diol and 2-amino-1-butanol. The diisocyanate was aliphatic hexamethylene diisocyanate. The polymers absorbed water in an amount that increased with the increasing content of the PEO segment in the polymer chain. The total amount of absorbed water did not exceed 2% for the poly(ester urethane)s and was as high as 212% for some poly(ester ether urethane)s that behaved in water like hydrogels. The polymers were subjected to in vitro degradation at 37 +/- 0.1 degrees C in phosphate buffer solutions for up to 76 weeks. The poly(ester urethane)s showed 1-2% mass loss at 48 weeks and 1.1-3.8% mass loss at 76 weeks. The poly(ester ether urethane)s manifested 1.6-76% mass loss at 48 weeks and 1.6-96% mass loss at 76 weeks. The increasing content and molecular weight of the PEO segment enhanced the rate of mass loss. Similar relations were also observed for polyurethanes from PEO-PPO-PEO (Pluronic) diols. Materials obtained with 2-amino-1-butanol as the chain extender degraded at a slower rate than similar materials synthesized with 1,4-butane diol. All the materials already manifested a progressive decrease in the molecular weight in the first month of in vitro aging. The rate of molecular weight loss was higher for poly(ester ether urethane)s than for poly(ester urethane)s. For poly(ester ether urethane)s, the rate of molecular weight loss was higher for materials containing Pluronic than for those containing PEO segments. All polymers calcified in vitro. The susceptibility to calcification increased with material hydrophilicity. The progressive deposition of calcium salt on the film surfaces resulted in the formation of large crystal aggregates, the structure of which depended on the chemical composition of the calcified material. Needle-like aggregates, resembling brushite, formed on the hydrophobic polyurethane, and plate-like crystals formed on the highly hydrophilic material. The calcium-to-phosphorus atomic ratio of the crystals growing on the samples was dependent on the chemical composition of the material and varied from 0.94 to 1.55.

Multidrug resistance (MDR) remains one of the biggest obstacles for effective cancer therapy. Currently there are only few methods that are available clinically that are used to bypass MDR with very limited success. In this review we describe how MDR can be overcome by a simple yet effective approach of using amphiphilic block copolymers. Triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), arranged in a triblock structure PEO-PPO-PEO, Pluronics or "poloxamers", raised a considerable interest in the drug delivery field. Previous studies demonstrated that Pluronics sensitize MDR cancer cells resulting in increased cytotoxic activity of Dox, paclitaxel, and other drugs by 2-3 orders of magnitude. Pluronics can also prevent the development of MDR in vitro and in vivo. Additionally, promising results of clinical studies of Dox/Pluronic formulation reinforced the need to ascertain a thorough understanding of Pluronic effects in tumors. These effects are extremely comprehensive and appear on the level of plasma membranes, mitochondria, and regulation of gene expression selectively in MDR cancer cells. Moreover, it has been demonstrated recently that Pluronics can effectively deplete tumorigenic intrinsically drug-resistant cancer stem cells (CSC). Interestingly, sensitization of MDR and inhibition of drug efflux transporters is not specific or selective to Pluronics. Other amphiphilic polymers have shown similar activities in various experimental models. This review summarizes recent advances of understanding the Pluronic effects in sensitization and prevention of MDR.

Dense, ultrathin networks of isocyanate terminated star-shaped poly(ethylene oxide) (PEO) molecules, cross-linked at their chain ends via urea groups, were shown to be extremely resistant to unspecific adsorption of proteins while at the same time suitable for easy biocompatible modification. Application by spin coating offers a simple procedure for the preparation of minimally interacting surfaces that are functionalized by suitable linker groups to immobilize proteins in their native conformations. These coatings form a versatile basis for biofunctional and biomimetic surfaces. We have demonstrated their advantageous properties by using single-molecule fluorescence microscopy to study immobilized proteins under destabilizing conditions. Biotinylated ribonuclease H (RNase H) was labeled with a fluorescence resonance energy transfer (FRET) pair of fluorescent dyes and attached to the surface by a biotin-streptavidin linkage. FRET analysis demonstrated completely reversible denaturation/renaturation behavior upon exposure of the surface-immobilized proteins to 6 M guanidinium chloride (GdmCl) followed by washing in buffer. A comparison with bovine serum albumin (BSA) coated surfaces and linear PEO brush surfaces yielded superior performance in terms of chemical stability, inertness and noninteracting nature of the star-polymer derived films.

Biomaterials-related infections pose serious problems in implant surgery, despite the development of non-adhesive coatings. Non-adhesive coatings, like polymer brush-coatings, have so far only been investigated with respect to preventing initial bacterial adhesion, but never with respect to effects on kinetics of bacterial growth. Here, we compare adhesion and 20 h growth of three bacterial strains (Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa) on pristine and brush-coated silicone rubber in a parallel plate flow chamber. Brush-coatings were made using a tri-block copolymer of polyethylene oxide (PEO) and polypropylene oxide (PPO). Brush-coatings prevented adhesion of staphylococci to below 5 x 10(5)cm(-2) after 30 min, which is a 10-fold reduction compared to pristine silicone rubber. Biofilms grew on both brush-coated and pristine silicone rubber, while the viability of biofilms on brush-coatings was higher than on pristine silicone rubber. However, biofilms on brush-coatings developed more slowly and detached almost fully by high fluid shear. Brush-coating remained non-adhesive after S. epidermidis biofilm formation and subsequent removal whereas a part of its functionality was lost after removal of S. aureus biofilms. Adhesion, growth and detachment of P. aeruginosa were not significantly different on brush-coatings as compared with pristine silicone rubber, although here too the viability of biofilms on brush-coatings was higher. We conclude that polymer brush-coatings strongly reduce initial adhesion of staphylococci and delay their biofilm growth. In addition, biofilms on brush-coatings are more viable and easily removed by the application of fluid shear.

Multiple deletions of mitochondrial DNA (mtDNA) are associated with different mitochondrial disorders inherited as autosomal dominant and recessive traits. Causative mutations have been found in five genes, mainly involved in mtDNA replication and stability. They include POLG1, the gene encoding the catalytic subunit of mtDNA polymerase (pol gamma), POLG2 encoding its accessory subunit, ANT1 coding the adenine nucleotide translocator and PEOPEOPEO). Genetic analysis revealed that 1) PEOPEO, since it accounts for 26.8% of familial cases, followed by ANT1 (14.6%) and POLG1 (9.8%); 2) no mutations in any of the known genes were found in 53.7% of probands of this series. Six novel missense mutations contributing to the mutational load of PEOPEO phenotype.

This paper summarizes recent advances in understanding the links between the cell's ability to maintain integrity of its mitochondrial genome and mitochondrial genetic diseases. Human mitochondrial DNA is replicated by the two-subunit DNA polymerase gamma (polgamma). We investigated the fidelity of DNA replication by polgamma with and without exonucleolytic proofreading and its p55 accessory subunit. Polgamma has high base substitution fidelity due to efficient base selection and exonucleolytic proofreading, but low frameshift fidelity when copying homopolymeric sequences longer than four nucleotides. Progressive external ophthalmoplegia (PEO) is a rare disease characterized by the accumulation of large deletions in mitochondrial DNA. Recently, several mutations in the polymerase and exonuclease domains of the human polgamma have been shown to be associated with PEO. We are analyzing the effect of these mutations on the human polgamma enzyme. In particular, three autosomal dominant mutations alter amino acids located within polymerase motif B of polgamma. These residues are highly conserved among family A DNA polymerases, which include T7 DNA polymerase and E.coli pol I. These PEO mutations have been generated in polgamma to analyze their effects on overall polymerase function as well as the effects on the fidelity of DNA synthesis. One mutation in particular, Y955C, was found in several families throughout Europe, including one Belgian family and five unrelated Italian families. The Y955C mutant polgamma retains a wild-type catalytic rate but suffers a 45-fold decrease in apparent binding affinity for the incoming dNTP. The Y955C derivative is also much less accurate than is wild-type polgamma, with error rates for certain mismatches elevated by 10- to 100-fold. The error prone DNA synthesis observed for the Y955C polgamma is consistent with the accumulation of mtDNA mutations in patients with PEO. The effects of other polgamma mutations associated with PEO are discussed.

Degradable polyurethane elastomers were synthesized using a diester chain extender. The chain extender was synthesized by a diesterification reaction between L-phenylalanine and 1,4-cyclohexane dimethanol to yield a diester, diamine. Soft segment chemistry (polycaprolactone diol, PCL and polyethylene oxide, PEO) and molecular weight were varied and the impact on polyurethane physicochemical and degradation characteristics was evaluated. It was found that the PEO containing polyurethanes absorbed large amounts of water while the PCL containing ones did not, indicating a large difference in bulk hydrophilicity. The rate of water vapor permeance (WVP) through the polyurethane films generally followed the water absorption trends. However, soft segment crystallinity, noted by DSC, for the PCL containing polyurethanes served to reduce WVP values with increasing PCL molecular weight. Polyurethane surface characterization was carried out by water contact angles and XPS. The PEO containing polyurethanes exhibited low contact angles in comparison with the PCL ones. In addition, angle-resolved XPS demonstrated soft segment surface enrichment in all cases typical for phase segregated materials. Significant variation in the physicochemical properties of the experimental polyurethanes was observed indicating potential use in a variety of biomaterials applications. An in vitro degradation study was carried out by incubating the polymers in 0.1 M TBS at 37 degrees C, pH 8.0 for up to 56 days. Degradation was followed by measuring mass loss, change in molecular weight by GPC and surface alteration by scanning electron microscopy. The polyurethane containing PEO was found to exhibit substantial mass and molecular weight loss over 56 days resulting in a porous material of little strength. In contrast, the PCL containing polyurethane displayed modest mass and molecular weight loss after 56 days. This polyurethane retained its strength and displayed little surface alteration after 56 days in buffer. It was hypothesized that differences in polyurethane hydrophilicity as well as initial molecular weight may have been responsible for the dramatic difference in degradation rate observed here.

The 3243A>> G mutation is one of the most frequently observed mutations of mitochondrial DNA (mtDNA), and is associated with numerous clinical presentations including mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), progressive external ophthalmoplegia (PEO) and diabetes and deafness. The routine diagnosis of the 3243A>> G mutation in blood is difficult as mutation levels are known to decrease in this tissue over time, while in some patients it may be absent. We have directly compared the levels of the 3243A>> G mutation in skeletal muscle, blood and urinary epithelial cells in 18 patients and observed a striking correlation between the mutation load in postmitotic muscle and urinary epithelium, a mitotic tissue. These data strongly support the use of urinary epithelial cells as the tissue of choice in the noninvasive diagnosis of the 3243A>> G mutation.

Polymerase gamma 1 (POLG) mutations are a frequent cause of both autosomal dominant and recessive complex neurological phenotypes. In contrast, only a single pathogenic mutation in one patient was reported in POLG2 so far. Here we describe a 62-year-old woman, carrying a novel heterozygous sequence variant in the POLG2 gene. She developed bilateral ptosis at 30 years of age, followed by exercise intolerance, muscle weakness and mild CK increase in her late forties. Muscle histology and respiratory chain activities were normal. Southern blot and long range PCR detected multiple mtDNA deletions, but no depletion in muscle DNA. Sequencing of POLG, PEOPEO.

Polyethylene oxide (PEO) of molecular weights 5,000, 10,000, 18,500, and 100,000 g/mol was covalently grafted to surfaces of otherwise cell adhesive polyethylene terephthalate (PET) films. Analysis of these surfaces by measurement of contact angles and ESCA verified the presence of the grafted PEO. Protein adsorption assays of radiolabeled albumin and fibrinogen showed a marked reduction in adsorbed protein for the 18,500 and 100,000 molecular weight PEO coupled surfaces. Cell growth assays using human foreskin fibroblasts in culture showed that the higher-molecular-weight PEO surfaces supported cell growth to a much lower extent than the two lower-molecular-weight PEOs. Flow of whole blood over these surfaces and visualization of platelet adherence using epifluorescence video-microscopy showed very low platelet adherence only on the two higher-molecular-weight PEO coupled surfaces. Scanning electron microscopy corroborated these results. It was concluded that PEO of molecular weights neighboring 18,500 and higher was effective in reducing protein adsorption and cellular interactions on these surfaces.

Hydrogels are very attractive delivery systems for hydrophilic macromolecules such as proteins and DNA because they provide a protective environment and allow control of diffusion by adjusting cross-link densities. Physically cross-linked hydrogels generated by rapid swelling upon exposure to an aqueous environment can be obtained from ABA triblock copolymers containing hydrophobic polyester A-blocks and hydrophilic polyether B-blocks. They provide an attractive alternative to chemically cross-linked systems since they allow incorporation of macromolecular drug substances under mild process conditions. Moreover, they show controlled degradation behavior and excellent biocompatibility. In this review the synthesis and characterization of ABA triblock copolymers from polyester hard segments and poly(ethylene oxide) [PEO] soft segments as well as their biological and degradation properties will be discussed. Their use as biodegradable drug delivery devices in the form of implants, micro- and nanospheres has attracted considerable interest especially for proteins and may provide an alternative to poly(lactide-co-glycolide).

The cellular internalization of polycaprolactone-b-poly(ethylene oxide) (PCL(20)-b-PEO(44)) copolymer micelles were investigated in PC12 cells cultures. The micelles were found to be internalized into PC12 cells when followed over the 4-h incubation period. Also, the internalization process was found to fulfill the basic criteria for endocytotic uptake in that it was time, temperature, pH and energy dependent. In addition, the use of other pharmacological manipulations (hypertonic treatment, Brefeldin A) provide further evidence that the mode of cellular internalization is in fact endocytotic.

The effect of differing densities of poly (ethylene glycol-2000) (PEG2000) at the particle surface of polystyrene-poly (ethylene glycol-2000) (PS-PEG2000) particles was assessed in terms of hydrophobic interaction chromatography (HIC) and the in vitro and in vivo behaviour of the particles. The particles, with different surface densities of PEG, were prepared by varying the copolymerizing reaction of styrene with a PEG macromonomer. There is a clear relationship between the surface density of PEG as determined by X-ray photoelectron spectroscopy and surface hydrophobicity as assessed by hydrophobic interaction chromatography (HIC). Similarly, the interaction of the particles with non-parenchymal liver cells in in vitro studies was shown to decrease as the surface density of PEG increases. The in vivo study investigating the biodistribution of the PS-PEG particles after intravenous injection into rats reveals that a relationship exists between the surface density of PEG and the extent to which the particles remain in the circulation, avoiding recognition by the reticuloendothelial system. Particles with the higher surface densities show increased circulatory times which compared well with data for particles prepared with the surface adsorbed PEO-PPO block copolymer, Poloxamine 908.

Recent studies suggest that immature, core-glycosylated DeltaF508-CFTR [the predominant mutant form of the CFTR (cystic fibrosis transmembrane conductance regulator)] can reach the plasma membrane under some conditions. In the present study we investigated this possibility since it has implications for understanding how therapeutics rescue the trafficking of mutant CFTR and perhaps other misfolded proteins. Core-glycosylated CFTR was labelled and pulled down on streptavidin beads after exposure to sulfo-NHS-SS-biotin [biotin attached to a reactive NHS (N-hydroxysuccinimide) ester with a disulfide spacer; molecular mass=606.7 Da]; however, intracellular proteins were also detected in the precipitates. When the R domain of CFTR was expressed in the cytosol of BHK (baby-hamster kidney) cells as a soluble polypeptide it was also labelled after surface biotinylation and pulled down on streptavidin beads. Intracellular biotinylation was reduced when cells were treated with sulfo-NHS-LC-biotin (biotin attached to a reactive NHS ester with an aminocaproic acid spacer) or sulfo-NHS-PEO(12)-biotin [biotin attached to a reactive NHS ester with a poly(ethylene glycol) spacer], but the reduction could be explained by the lower reactivity of these reagents. The R domain was detected on Western blots after loading <0.25% of the pulldown sample ( approximately 0.01% of total lysate protein), a fraction that could be ascribed to cells that were permeable to ethidium homodimer-1 (molecular mass=856.8 Da) and propidium iodide (molecular mass=668.6 Da). When BHK cells were incubated at 29 degrees C to rescue DeltaF508-CFTR trafficking, and then biotinylated and sorted to remove permeable cells, labelling of core-glycosylated DeltaF508-CFTR was no longer detected although a weak signal was still observed using CFBE (cystic fibrosis bronchial epithelial) cells. These results suggest that there is weak surface expression of immature DeltaF508-CFTR on airway epithelial cells and demonstrate the need to remove permeable cells when studying CFTR glycoforms by surface biotinylation.