Progressive external ophthalmoplegias (PEO) characterized by accumulation of large-scale mitochondrial DNA (mtDNA) deletions are rare human diseases. We mapped a new locus for dominant PEO at 15q22-q26 in a Belgian pedigree and identified a heterozygous mutation (Y955C) in the polymerase motif B of the mtDNA polymerase gamma (POLG). We identified three additional POLG missense mutations compatible with recessive PEO In two nuclear families. POLG is the only DNA polymerase responsible for mtDNA replication.

Silk fibroin fiber scaffolds containing bone morphogenetic protein 2 (BMP-2) and/or nanoparticles of hydroxyapatite (nHAP) prepared via electrospinning were used for in vitro bone formation from human bone marrow-derived mesenchymal stem cells (hMSCs). BMP-2 survived the aqueous-based electrospinnig process in bioactive form. hMSCs were cultured for up to 31 days under static conditions in osteogenic media on the scaffolds (silk/PEO/BMP-2, silk/PEO/nHAP, silk/PEO/nHAP/BMP-2) and controls (silk/PEO, silk/PEO extracted). Electrospun silk fibroin-based scaffolds supported hMSC growth and differentiation toward osteogenic outcomes. The scaffolds with the co-processed BMP-2 supported higher calcium deposition and enhanced transcript levels of bone-specific markers than in the controls, indicating that these nanofibrous electrospun silk scaffolds were an efficient delivery system for BMP-2. X-ray diffraction (XRD) analysis revealed that the apatite formed on the silk fibroin/BMP-2 scaffolds had higher crystallinity than on the silk fibroin scaffold controls. In addition, nHAP particles were incorporated into the electrospun fibrous scaffolds during processing and improved bone formation. The coexistence of BMP-2 and nHAP in the electrospun silk fibroin fibers resulted in the highest calcium deposition and upregulation of BMP-2 transcript levels when compared with the other systems. The results suggest that electrospun silk-fibroin-based scaffolds are potential candidates for bone tissue engineering. Furthermore, the mild aqueous process required to spin the fibers offers an important option for delivery of labile cytokines and other components into the system.

Dendrimers are highly branched macromolecules of low polydispersity that provide many exciting opportunities for design of novel drug-carriers, gene delivery systems and imaging agents. They hold promise in tissue targeting applications, controlled drug release and moreover, their interesting nanoscopic architecture might allow easier passage across biological barriers by transcytosis. However, from the vast array of structures currently emerging from synthetic chemistry it is essential to design molecules that have real potential for in vivo biological use. Here, polyamidoamine (PAMAM, Starburst), poly(propyleneimine) with either diaminobutane or diaminoethane as core, and poly(ethylene oxide) (PEO) grafted carbosilane (CSi-PEO) dendrimers were used to study systematically the effect of dendrimer generation and surface functionality on biological properties in vitro. Generally, dendrimers bearing -NH(2) termini displayed concentration- and in the case of PAMAM dendrimers generation-dependent haemolysis, and changes in red cell morphology were observed after 1 h even at low concentrations (10 microg/ml). At concentrations below 1 mg/ml CSi-PEO dendrimers and those dendrimers with carboxylate (COONa) terminal groups were neither haemolytic nor cytotoxic towards a panel of cell lines in vitro. In general, cationic dendrimers were cytotoxic (72 h incubation), displaying IC(50) values=50-300 microg/ml dependent on dendrimer-type, cell-type and generation. Preliminary studies with polyether dendrimers prepared by the convergent route showed that dendrimers with carboxylate and malonate surfaces were not haemolytic at 1 h, but after 24 h, unlike anionic PAMAM dendrimers they were lytic. Cationic 125I-labelled PAMAM dendrimers (gen 3 and 4) administered intravenously (i.v.) to Wistar rats ( approximately 10 microg/ml) were cleared rapidly from the circulation (<2% recovered dose in blood at 1 h). Anionic PAMAM dendrimers (gen 2.5, 3.5 and 5.5) showed longer circulation times ( approximately 20-40% recovered dose in blood at 1 h) with generation-dependent clearance rates; lower generations circulated longer. For both anionic and cationic species blood levels at 1 h correlated with the extent of liver capture observed (30-90% recovered dose at 1 h). 125I-Labelled PAMAM dendrimers injected intraperitoneally were transferred to the bloodstream within an hour and their subsequent biodistribution mirrored that seen following i.v. injection. Inherent toxicity would suggest it unlikely that higher generation cationic dendrimers will be suitable for parenteral administration, especially if they are to be used at a high dose. In addition it is clear that dendrimer structure must also be carefully tailored to avoid rapid hepatic uptake if targeting elsewhere (e.g. tumour targeting) is a primary objective.

Mutations in nuclear genes involved in mitochondrial DNA (mtDNA) maintenance cause a wide range of clinical phenotypes associated with the secondary accumulation of multiple mtDNA deletions in affected tissues. The majority of families with autosomal dominant progressive external ophthalmoplegia (<em>PEO</em>) harbour mutations in genes encoding one of three well-characterized proteins--pol gamma, Twinkle or Ant 1. Here we show that a heterozygous mis-sense mutation in OPA1 leads to multiple mtDNA deletions in skeletal muscle and a mosaic defect of cytochrome c oxidase (COX). The disorder presented with visual failure and optic atrophy in childhood, followed by <em>PEO</em>, ataxia, deafness and a sensory-motor neuropathy in adult life. COX-deficient skeletal muscle fibres contained supra-threshold levels of multiple mtDNA deletions, and genetic linkage, sequencing and expression analysis excluded POLG1, <em>PEO</em>1 and SLC25A4, the gene encoding Ant 1, as the cause. This demonstrates the importance of OPA1 in mtDNA maintenance, and implicates OPA1 in diseases associated with secondary defects of mtDNA.

Aligned electrospun scaffolds are promising tools for engineering fibrous musculoskeletal tissues, as they reproduce the mechanical anisotropy of these tissues and can direct ordered neo-tissue formation. However, these scaffolds suffer from a slow cellular infiltration rate, likely due in part to their dense fiber packing. We hypothesized that cell ingress could be expedited in scaffolds by increasing porosity, while at the same time preserving overall scaffold anisotropy. To test this hypothesis, poly(epsilon-caprolactone) (a slow-degrading polyester) and poly(ethylene oxide) (a water-soluble polymer) were co-electrospun from two separate spinnerets to form dual-polymer composite fiber-aligned scaffolds. Adjusting fabrication parameters produced aligned scaffolds with a full range of sacrificial (PEO) fiber contents. Tensile properties of scaffolds were functions of the ratio of PCL to PEO in the composite scaffolds, and were altered in a predictable fashion with removal of the PEO component. When seeded with mesenchymal stem cells (MSCs), increases in the starting sacrificial fraction (and porosity) improved cell infiltration and distribution after three weeks in culture. In pure PCL scaffolds, cells lined the scaffold periphery, while scaffolds containing >50% sacrificial PEO content had cells present throughout the scaffold. These findings indicate that cell infiltration can be expedited in dense fibrous assemblies with the removal of sacrificial fibers. This strategy may enhance in vitro and in vivo formation and maturation of functional constructs for fibrous tissue engineering.

Kearns-Sayre syndrome (KSS) and progressive external ophthalmoplegia (PEO) are related neuromuscular disorders characterized by ocular myopathy and ophthalmoplegia. Almost all patients with KSS and about half with PEO harbor large deletions in their mitochondrial genomes. The deletions differ in both size and location, except for one, 5 kilobases long, that is found in more than one-third of all patients examined. This common deletion was found to be flanked by a perfect 13-base pair direct repeat in the normal mitochondrial genome. This result suggests that homologous recombination deleting large regions of intervening mitochondrial DNA, which previously had been observed only in lower eukaryotes and plants, operates in mammalian mitochondrial genomes as well, and is at least one cause of the deletions found in these two related mitochondrial myopathies.

Mitochondrial genetic diseases can result from defects in mitochondrial DNA (mtDNA) in the form of deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These mutations may be spontaneous, maternally inherited, or a result of inherited nuclear defects in genes that maintain mtDNA. This review focuses on our current understanding of nuclear gene mutations that produce mtDNA alterations and cause mitochondrial depletion syndrome (MDS), progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). To date, all of these etiologic nuclear genes fall into one of two categories: genes whose products function directly at the mtDNA replication fork, such as POLG, POLG2, and TWINKLE, or genes whose products supply the mitochondria with deoxynucleotide triphosphate pools needed for DNA replication, such as TK2, DGUOK, TP, SUCLA2, ANT1, and possibly the newly identified MPV17.

BACKGROUND

With the thrombogenic tendency and permanent implant nature of metallic stents, synthetic polymers have been proposed as candidate materials for stents and local drug delivery designs. We investigated the biocompatibility of several synthetic polymers after experimental placement in the coronary artery.

RESULTS

Five different biodegradable polymers (polyglycolic acid/polylactic acid [PGLA], polycaprolactone [PCL], polyhydroxybutyrate valerate [PHBV], polyorthoester [POE], and polyethyleneoxide/polybutylene terephthalate [PEO/ PBTP]) and three nonbiodegradable polymers (polyurethane [PUR], silicone [SIL], and polyethylene terephthalate [PETP]) were tested as strips deployed longitudinally across 90 degrees of the circumferential surface of coil wire stents. Appropriately sized polymer-loaded stents were implanted in porcine coronary arteries of 2.5- to 3.0-mm diameter. Four weeks after implantation, stent patency was assessed by angiography followed by microscopic examination of the coronary arteries. The biodegradable PCL, PHBV, and POE and the nonbiodegradable PUR and SIL evoked extensive inflammatory responses and fibrocellular proliferation (thickness of tissue response: 0.79 +/- 0.22, 1.12 +/- 0.01, 2.36 +/- 0.60, 1.24 +/- 0.36, and 1.43 +/- 0.15 mm, respectively). Less but still severe responses were observed for the biodegradable PGLA and PEO/PBTP (0.46 +/- 0.18 and 0.61 +/- 0.23 mm, respectively) and for the nonbiodegradable PETP (0.46 +/- 0.11 mm).

CONCLUSIONS

An array of both biodegradable and nonbiodegradable polymers has been demonstrated to induce a marked inflammatory reaction within the coronary artery with subsequent neointimal thickening, which was not expected on the basis of in vitro tests. The observed tissue response may be attributable to a combination of parent polymer compound, biodegradation products, and possibly implant geometry.

Control of silk fibroin concentration in aqueous solutions via osmotic stress was studied to assess relationships to gel formation and structural, morphological, and functional (mechanical) changes associated with this process. Environmental factors potentially important in the in vivo processing of aqueous silk fibroin were also studied to determine their contributions to this process. Gelation of silk fibroin aqueous solutions was affected by temperature, Ca(2+), pH, and poly(ethylene oxide) (PEO). Gelation time decreased with increase in protein concentration, decrease in pH, increase in temperature, addition of Ca(2+), and addition of PEO. No change of gelation time was observed with the addition of K(+). Upon gelation, a random coil structure of the silk fibroin was transformed into a beta-sheet structure. Hydrogels with fibroin concentrations >4 wt % exhibited network and spongelike structures on the basis of scanning electron microscopy. Pore sizes of the freeze-dried hydrogels were smaller as the silk fibroin concentration or gelation temperature was increased. Freeze-dried hydrogels formed in the presence of Ca(2+) exhibited larger pores as the concentration of this ion was increased. Mechanical compressive strength and modulus of the hydrogels increased with increase in protein concentration and gelation temperature. The results of these studies provide insight into the sol-gel transitions that silk fibroin undergoes in glands during aqueous processing while also providing important insight in the in vitro processing of these proteins into useful new materials.

Defects of mitochondrial DNA (mtDNA) maintenance have recently been associated with inherited neurodegenerative and muscle diseases and the aging process. Twinkle is a nuclear-encoded mtDNA helicase, dominant mutations of which cause adult-onset progressive external ophthalmoplegia (PEO) with multiple mtDNA deletions. We have generated transgenic mice expressing mouse Twinkle with PEO patient mutations. Multiple mtDNA deletions accumulate in the tissues of these mice, resulting in progressive respiratory dysfunction and chronic late-onset mitochondrial disease starting at 1 year of age. The muscles of the mice faithfully replicate all of the key histological, genetic, and biochemical features of PEO patients. Furthermore, the mice have progressive deficiency of cytochrome c oxidase in distinct neuronal populations. These "deletor" mice do not, however, show premature aging, indicating that subtle accumulation of mtDNA deletions and progressive respiratory chain dysfunction are not sufficient to accelerate aging. This model is a valuable tool for therapy development and testing for adult-onset mitochondrial disorders.

A new family of nanoscale materials on the basis of dispersed networks of cross-linked ionic and nonionic hydrophilic polymers is being developed. One example is the nanosized cationic network of cross-linked poly(ethylene oxide) (PEO) and polyethyleneimine (PEI), PEO-cl-PEI nanogel. Interaction of anionic amphiphilic molecules or oligonucleotides with PEO-cl-PEI results in formation of nanocomposite materials in which the hydrophobic regions from polyion-complexes are joined by the hydrophilic PEO chains. Formation of polyion-complexes leads to the collapse of the dispersed gel particles. However, the complexes form stable aqueous dispersions due to the stabilizing effect of the PEO chain. These systems allow for immobilization of negatively charged biologically active compounds such as retinoic acid, indomethacin and oligonucleotides (bound to polycation chains) or hydrophobic molecules (incorporated into nonpolar regions of polyion-surfactant complexes). The nanogel particles carrying biological active compounds have been modified with polypeptide ligands to enhance receptor-mediated delivery. Efficient cellular uptake and intracellular release of oligonucleotides immobilized in PEO-cl-PEI nanogel have been demonstrated. Antisense activity of an oligonucleotide in a cell model was elevated as a result of formulation of oligonucleotide with the nanogel. This delivery system has a potential of enhancing oral and brain bioavailability of oligonucleotides as demonstrated using polarized epithelial and brain microvessel endothelial cell monolayers.

The success of anti-cancer therapies largely depends on the ability of the therapeutics to reach their designated cellular and intracellular target sites, while minimizing accumulation and action at non-specific sites. Surface modification of nanoparticulate carriers with poly(ethylene glycol) (PEG)/poly(ethylene oxide) (PEO) has emerged as a strategy to enhance solubility of hydrophobic drugs, prolong circulation time, minimize non-specific uptake, and allow for specific tumor-targeting through the enhanced permeability and retention effect. Furthermore, PEG/PEO modification has emerged as a platform for incorporation of active targeting ligands, thereby providing the drug and gene carriers with specific tumor-targeting properties through a flexible tether. This review focuses on the recent developments surrounding such PEG/PEO-surface modification of polymeric nanocarriers to promote tumor-targeting capabilities, thereby enhancing efficacy of anti-cancer therapeutic strategies.

Large-scale deletions of mitochondrial DNA (mtDNA) have been described in patients with progressive external ophthalmoplegia (PEO) and ragged red fibers. We have determined the exact deletion breakpoint in 28 cases with PEO, including 12 patients already shown to harbor an identical deletion; the other patients had 16 different deletions. The deletions fell into two classes. In Class I (9 deletions; 71% of the patients), the deletion was flanked by perfect direct repeats, located (in normal mtDNA) at the edges of the deletion. In Class II (8 deletions; 29% of patients), the deletions were not flanked by any obviously unique repeat element, or they were flanked by repeat elements which were located imprecisely relative to the breakpoints. Computer analysis showed a correlation between the location of the deletion breakpoints and sequences in human mtDNA similar to the target sequence for Drosophila topoisomerase II. It is not known how these deletions originate, but both slipped mispairing and legitimate recombination could be mechanisms playing a major role in the generation of the large mtDNA deletions found in PEO.

Electrospinning for the formation of nanoscale diameter fibers has been explored for high-performance filters and biomaterial scaffolds for vascular grafts or wound dressings. Fibers with nanoscale diameters provide benefits due to high surface area. In the present study we explore electrospinning for protein-based biomaterials to fabricate scaffolds and membranes from regenerated silkworm silk, Bombyx mori, solutions. To improve processability of the protein solution, poly(ethylene oxide) (PEO) with molecular weight of 900,000 was blended with the silk fibroin. A variety of compositions of the silk/PEO aqueous blends were successfully electrospun. The morphology of the fibers was characterized using high-resolution scanning electron microscopy. Fiber diameters were uniform and less than 800 nm. The composition was estimated by X-ray photoelectron spectroscopy to characterize silk/PEO surface content. Aqueous-based electrospining of silk and silk/PEO blends provides potentially useful options for the fabrication of biomaterial scaffolds based on this unique fibrous protein.

Meshes of collagen and/or elastin were successfully prepared by means of electrospinning from aqueous solutions. Flow rate, applied electric field, collecting distance and composition of the starting solutions determined the morphology of the obtained fibres. Addition of PEO (M(w)=8 x 10(6)) and NaCl was always necessary to spin continuous and homogeneous fibres. Spinning a mixture of collagen and elastin resulted in fibres in which the single components could not be distinguished by SEM. Increasing the elastin content determined an increase in fibres diameters from 220 to 600 nm. The voltage necessary for a continuous production of fibres was dependent on the composition of the starting solution, but always between 10 and 25 kV. Under these conditions, non-woven meshes could be formed and a partial orientation of the fibres constituting the mesh was obtained by using a rotating tubular mandrel as collector. Collagen/elastin (1:1) meshes were stabilized by crosslinking with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). This treatment afforded materials with a high thermal stability (T(d)=79 degrees C) without altering their original morphology. Upon crosslinking PEO and NaCl were fully leached out. Smooth muscle cells grew as a confluent layer on top of the crosslinked meshes after 14 d of culture.

In this article we report on the development of polymeric micelles that can integrate multiple functions in one system, including the capability to accommodate a combination of therapeutic entities with different physicochemical properties (i.e., siRNA and doxorubicin; DOX), passive and active cancer targeting, cell membrane translocation, and pH-triggered drug release. A micellar system was constructed from degradable poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) block copolymers with functional groups on both blocks. The functional group on the PCL block was used to incorporate short polyamines for complexation with siRNA or to chemically conjugate DOX via a pH-sensitive hydrazone linkage. A virus mimetic shell was conferred by attaching two ligands, i.e., the integrin αvβ3-specific ligand (RGD4C) for active cancer targeting and the cell-penetrating peptide TAT for membrane activity. This system was used to improve the efficacy of DOX in multidrug-resistant MDA-MB-435 human tumor models that overexpress P-glycoprotein (P-gp), by simultaneous intracellular delivery of DOX and siRNA against P-gp expression. The carrier was tagged with near-infrared fluorescent imaging probes to provide a means to follow the fate of the system in vivo upon intravenous administration. Dy677-labeled siRNA was also used to assess the in vivo stability of the siRNA carrier. This multifunctional polymeric micellar system was shown to be capable of DOX and siRNA delivery to their intracellular targets, leading to the inhibition of P-gp-mediated DOX resistance in vitro and targeting of αvβ3-positive tumors in vivo.

One form of familial progressive external ophthalmoplegia with multiple mitochondrial DNA deletions recently has been associated with mutations in POLG1, the gene encoding pol gammaA, the catalytic subunit of mitochondrial DNA polymerase. We screened the POLG1 gene in several PEO families and identified five different heterozygous missense mutations of POLG1 in 10 autosomal dominant families. Recessive mutations were found in three families. Our data show that mutations of POLG1 are the most frequent cause of familial progressive external ophthalmoplegia associated with accumulation of multiple mitochondrial DNA deletions, accounting for approximately 45% of our family cohort.

OBJECTIVE

The aim of this study was to investigate the interaction between the components of novel chitosan (CS) and CS/ethylene oxide-propylene oxide block copolymer (PEO-PPO) nanoparticles and to evaluate their potential for the association and controlled release of proteins and vaccines.

METHODS

The presence of PEO-PPO on the surface of the nanoparticles and its interaction with the CS was identified by X-ray photoelectron spectroscopy (XPS). The mechanism of protein association was elucidated using several proteins, bovine serum albumin (BSA), and tetanus and diphtheria toxoids, and varying the formulation conditions (different pH values and concentrations of PEO-PPO), and the stage of protein incorporation into the nanoparticles formation medium.

RESULTS

BSA and tetanus and diphtheria toxoids were highly associated with CS nanoparticles partly due to electrostatic interactions between the carboxyl groups of the protein and the amine groups of CS. PEO-PPO also interacted electrostatically with CS, thus competing with the proteins for association with CS nanoparticles. A visible amount of PEO-PPO was projected towards the outer phase of the nanoparticles. Proteins were released from the nanoparticles at an almost constant rate, the intensity of which was closely related to the protein loading. Furthermore, the tetanus vaccine was released in the active form for at least 15 days.

CONCLUSIONS

CS and CS/PEO-PPO nanoparticles prepared by a very mild ionic crosslinking technique are novel and suitable systems for the entrapment and controlled release of proteins and vaccines.

Chitosan-based nanofibers with an average fiber diameter controllable from a few microns down to approximately 40 nm and a narrow size distribution were fabricated by electrospinning solutions containing chitosan, polyethylene oxide (PEO), and Triton X-100. Rheological study showed a strong dependence of spinnability and fiber morphology on solution viscosity and thus on chitosan-to-PEO ratio. The nanofibers can be deposited either as a nonwoven mat or as a highly aligned bundle of controllable size. Potential use of this nanofibrous matrix for tissue engineering was studied by examining its integrity in water and cellular compatibility. It was found that the matrix with a chitosan/PEO ratio of 90/10 retained excellent integrity of the fibrous structure in water. Experimental results from cell stain assay and SEM imaging showed that the nanofibrous structure promoted the attachment of human osteoblasts and chondrocytes and maintained characteristic cell morphology and viability throughout the period of study. This nanofibrous matrix is of particular interest in tissue engineering for controlled drug release and tissue remodeling.

Block copolymer micelles encapsulate water insoluble drugs by chemical and physical means, and they may target therapeutics to their site of action in a passive or active way. In this review, we focus on micelles self-assembled from poly(ethylene oxide)-block-poly(L-amino acid) (PEO-b-PLAA). A common theme in these studies is the chemical modification of the core-forming PLAA block used to adjust and optimize the properties of PEO-b-PLAA micelles for drug delivery. Micelle-forming block copolymer-drug conjugates, micellar nanocontainers and polyion complex micelles have been obtained that mimic functional aspects of biological carriers, namely, lipoproteins and viruses. PEO-b-PLAA micelles may be advantageous in terms of safety, stability, and scale-up.

The expression of neutrophil gelatinase-associated lipocalin (NGAL) has been shown to be upregulated in ovarian cancer cells. In this study, we report that the expression of immunoreactive NGAL (irNGAL) in ovarian tumors changes with disease grade and that this change is reflected in the concentration of NGAL in peripheral blood. A total of 59 ovarian tissues including normal, benign, borderline malignant and grades 1, 2 and 3 malignant were analyzed using immunohistochemistry. irNGAL was not present in normal ovaries and the NGAL expression was weak to moderate in benign tissues. Both borderline and grade 1 tumors displayed the highest amount of NGAL expression with moderate to strong staining, whereas in grade 2 and 3 tumors, the extent of staining was significantly less (p < 0.01) and staining intensity was weak to moderate. Staining in all cases was confined to the epithelium. NGAL expression was analyzed by ELISA in 62 serum specimens from normal and different grades of cancer patients. Compared to control samples, the NGAL concentration was 2 and 2.6-fold higher in the serum of patients with benign tumors and cancer patients with grade 1 tumors (p < 0.05) and that result was consistent with the expression of NGAL performed by Western blot. NGAL expression was evaluated by Western blot in an immortalized normal ovarian cell line (IOSE29) as well as ovarian cancer cell lines. Moderate to strong expression of NGAL was observed in epithelial ovarian cancer cell lines SKOV3 and OVCA433 while no expression of NGAL was evident in normal IOSE29 and mesenchyme-like OVHS1, PEO.36 and HEY cell lines. NGAL expression was downregulated in ovarian cancer cell lines undergoing epithelio-mesenchymal transition (EMT) induced by epidermal growth factor (EGF). Downregulation of NGAL expression correlated with the upregulation of vimentin expression, enhanced cell dispersion and downregulation of E-cadherin expression, some of the hallmarks of EMT. EGF-induced EMT phenotypes were inhibited in the presence of AG1478, an inhibitor of EGF receptor tyrosine kinase activity. These data indicate that NGAL may be a good marker to monitor changes of benign to premalignant and malignant ovarian tumors and that the molecule may be involved in the progression of epithelial ovarian malignancies.

Amphiphilic compounds such as lipids and surfactants are fundamental building blocks of soft matter. We describe experiments with poly(1,2-butadiene-b-ethylene oxide) (PB-PEO) diblock copolymers, which form Y-junctions and three-dimensional networks in water at weight fractions of PEOintermediate to those associated with vesicle and wormlike micelle morphologies. Fragmentation of the network produces a nonergodic array of complex reticulated particles that have been imaged by cryogenic transmission electron microscopy. Data obtained with two sets of PB-PEOcompounds indicate that this type of self-assembly appears above a critical molecular weight. These block copolymers represent versatile amphiphiles, mimicking certain low molecular weight three-component (surfactant/water/oil) microemulsions, without addition of a separate hydrophobe.

A biodegradable positron-emitting dendritic nanoprobe targeted at alpha(v)beta(3) integrin, a biological marker known to modulate angiogenesis, was developed for the noninvasive imaging of angiogenesis. The nanoprobe has a modular multivalent core-shell architecture consisting of a biodegradable heterobifunctional dendritic core chemoselectively functionalized with heterobifunctional polyethylene oxide (PEO) chains that form a protective shell, which imparts biological stealth and dictates the pharmacokinetics. Each of the 8 branches of the dendritic core was functionalized for labeling with radiohalogens. Placement of radioactive moieties at the core was designed to prevent in vivo dehalogenation, a potential problem for radiohalogens in imaging and therapy. Targeting peptides of cyclic arginine-glycine-aspartic acid (RGD) motifs were installed at the terminal ends of the PEO chains to enhance their accessibility to alpha(v)beta(3) integrin receptors. This nanoscale design enabled a 50-fold enhancement of the binding affinity to alpha(v)beta(3) integrin receptors with respect to the monovalent RGD peptide alone, from 10.40 nM to 0.18 nM IC(50). Cell-based assays of the (125)I-labeled dendritic nanoprobes using alpha(v)beta(3)-positive cells showed a 6-fold increase in alpha(v)beta(3) receptor-mediated endocytosis of the targeted nanoprobe compared with the nontargeted nanoprobe, whereas alpha(v)beta(3)-negative cells showed no enhancement of cell uptake over time. In vivo biodistribution studies of (76)Br-labeled dendritic nanoprobes showed excellent bioavailability for the targeted and nontargeted nanoprobes. In vivo studies in a murine hindlimb ischemia model for angiogenesis revealed high specific accumulation of (76)Br-labeled dendritic nanoprobes targeted at alpha(v)beta(3) integrins in angiogenic muscles, allowing highly selective imaging of this critically important process.

Progressive external ophthalmoplegia (PEO) is a mitochondrial disorder associated with mutations in the POLG gene encoding the mitochondrial DNA polymerase (pol gamma). Four autosomal dominant mutations that cause PEO encode the amino acid substitutions G923D, R943H, Y955C and A957S in the polymerase domain of pol gamma. A homology model of the pol gamma catalytic domain in complex with DNA was developed to investigate the effects of these mutations. Two mutations causing the most severe disease phenotype, Y955C and R943H, change residues that directly interact with the incoming dNTP. Polymerase mutants exhibit 0.03-30% wild-type polymerase activity and a 2- to 35-fold decrease in nucleotide selectivity in vitro. The reduced selectivity and catalytic efficiency of the autosomal dominant PEO mutants predict in vivo dysfunction, and the extent of biochemical defects correlates with the clinical severity of the disease.