A single-step synthesis of gold nanoparticles with an average diameter of approximately 10 nm from hydrogen tetrachloroaureate(III) hydrate (HAuCl4.3H2O) has been achieved in air-saturated aqueous solutions that contain poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymers but not any other reducing agent. These amphiphilic block copolymers act as both reductants and colloidal stabilizers and prove very efficient in both functions. The formation of gold nanoparticles is controlled by the overall molecular weight and relative block length of the block copolymer. The synthesis procedure reported here is environmentally benign and economic, as it involves the minimum possible number of components: it uses water as the solvent, it uses commercially available polymers, it proceeds fast to completion, and it results in a "ready-to-use" product.

Polyethylene oxide (PEO) surfaces were prepared by the addition of PEO-containing amphiphilic block copolymers as surface modifying additives and of dicumyl peroxide (DCP) as a crosslinking agent in segmented polyurethane (PU). PEO-polypropylene oxide-PEO triblock copolymers (Pluronics) with different PEO chain length (from 0 to 98) were used as the surface modifying additives. The PEO additives in the PU film were then crosslinked to be stably entrapped in the PU matrix. The crosslinking was done by free radicals produced from the decomposition of DCP in the film through heating (120 degrees C) or ultraviolet irradiation (254 nm). The surface properties of the PEO additive-entrapped PU films were investigated by the measurement of water contact angles and electron spectroscopy for chemical analysis. The bulk properties such as water absorption, long-term film stability, and tensile strength and elongation at break, were also investigated. It was observed that addition of a small amount (5 wt% based on PU) of the PEO additives resulted in a considerable change of surface characteristics. The PEO additives were stably entrapped in the PU films by crosslinking of them, without significant changes of bulk properties of the films. From the platelet adhesion test on the prepared PEO additive-containing film surfaces, it was observed that the platelet adhesion on the surfaces decreases with increase in PEO chain length of PEO additives. The film surface containing additive with long PEO chains (chain length of 98) was particularly effective in preventing platelet adhesion. The crosslinking of the PEO additives in PU films did not affect the behavior of platelet adhesion on the surfaces; the films with crosslinked PEO additives showed similar platelet adhesion on the surfaces to the films with uncrosslinked ones.

Polyurethanes (PU) were synthesized from 4,4'-diphenylmethane diisocyanate and polytetramethylene glycol, and subsequently with ethylene diamine as a chain extender. The PU film was exposed to oxygen plasma glow discharge to produce peroxides on the surfaces. These peroxides were then used as catalysts for the copolymerization of acrylic acid (AA) and methyl acrylate (MA) in order to prepare carboxyl group-introduced PU (PU-C). Heparin-immobilized PU was prepared using the coupling reaction of PU-C with polyethylene oxide (PEO) followed by the reaction of grafted PEO with heparin. The surface-modified PUs were then characterized by attenuated total reflection Fourier transform infrared spectroscopy, electron spectroscopy for chemical analysis (ESCA), and a contact angle goniometer. The concentration of carboxylic acid groups on the PU surfaces could be controlled within the range of 0.47-1.68 micromol cm(-2) by the copolymerization of AA and MA. The amounts of heparin coupled to terminus amino groups on PU-6 and PU-33 were 1.30 and 1.16 microg cm(-2), respectively. The water contact angle of the PU was decreased by AA grafting, and further decreased by PEO grafting and heparin immobilization, showing an increased hydrophilicity of the modified PUs. A 3% loss from the originally bound heparin appeared within several hours and thereafter almost no heparin was released when heparin-immobilized PUs were immersed in a physiological solution for 100 h, indicating the covalent immobilization of heparin on the surfaces.

BACKGROUND

The POLG1 gene encodes the catalytic subunit of DNA polymerase gamma, essential for mitochondrial DNA replication and repair. Mutations in POLG1 have been linked to a spectrum of clinical phenotypes, and may account for up to 25% of all adult presentations of mitochondrial disease.

RESULTS

We present 14 patients, with characteristic features of mitochondrial disease including progressive external ophthalmoplegia (PEO) and Alpers-Huttenlocher syndrome and laboratory findings indicative of mitochondrial dysfunction, including cytochrome c oxidase (COX) deficiency and multiple deletions or depletion of the mitochondrial DNA. Four novel POLG1 missense substitutions (p.R597W, p.L605R, p.G746S, p.A862T), are described, together with the first adult patient with a recently described polymerase domain mutation (p.R1047W). All novel changes were rare in a control population and affected highly conserved amino acids.

CONCLUSIONS

The addition of these substitutions-including the first report of a dinucleotide mutation (c.1814_1815TT>GC)-to the growing list of defects further confirms the importance of POLG1 mutations as the underlying abnormality in a range of neurological presentations.

Muscle biopsy provides the best tissue to confirm a mitochondrial cytopathy. Histochemical features often correlate with specific syndromes and facilitate the selection of biochemical and genetic studies. Ragged-red fibres nearly always indicate a combination defect of respiratory complexes I and IV. Increased punctate lipid within myofibers is a regular feature of Kearns-Sayre and PEO, but not of MELAS and MERRF. Total deficiency of succinate dehydrogenase indicates a severe defect in Complex II; total absence of cytochrome-c-oxidase activity in all myofibres correlates with a severe deficiency of Complex IV or of coenzyme-Q10. The selective loss of cytochrome-c-oxidase activity in scattered myofibers, particularly if accompanied by strong succinate dehydrogenase staining in these same fibres, is good evidence of mitochondrial cytopathy and often of a significant mtDNA mutation, though not specific for Complex IV disorders. Glycogen may be excessive in ragged-red zones. Ultrastructure provides morphological evidence of mitochondrial cytopathy, in axons and endothelial cells as well as myocytes. Abnormal axonal mitochondria may contribute to neurogenic atrophy of muscle, a secondary chronic feature. Quantitative determinations of respiratory chain enzyme complexes, with citrate synthase as an internal control, confirm the histochemical impressions or may be the only evidence of mitochondrial disease. Biological and technical artifacts may yield falsely low enzymatic activities. Genetic studies screen common point mutations in mtDNA. The brain exhibits characteristic histopathological alterations in mitochondrial diseases. Skin biopsy is useful for mitochondrial ultrastructure in smooth erector pili muscles and axons; skin fibroblasts may be grown in culture. Mitochondrial alterations occur in many nonmitochondrial diseases and also may be induced by drugs and toxins.

Since the early days of Darwin, monocot coleoptiles have been used to investigate indole-3-acetic acid (IAA) production, polar transport and tropisms. Here, using maize coleoptiles, we first showed that polar transport of IAA synthesized at the tip region is regulated by ZmPIN(s). Then, the TIR/AFBs-mediated auxin signaling pathway corresponds to the asymmetric IAA flow after gravi-stimulus, which results in tropic curvature. When [(13)C(11)(15)N(2)]Trp was applied to coleoptile tips, substantial amounts of the stable isotope were incorporated into IAA at the tip region, and the labeled IAA was transported in a polar manner at approximately 7 mm h(-1). Immunohistochemical analyses revealed that ZmPIN1(s) was present in almost all cells. ZmPIN1(s) showed a relatively non-polar distribution at the tip, but a basal cellular localization at lower regions. Application of the IAA transport inhibitors 1-N-naphthylphthalamic acid (NPA) and brefeldin A (BFA) at the very tip region almost completely inhibited IAA movement from the tip. These inhibitors also severely suppressed gravitropic bending. PEO-IAA, an auxin antagonist that binds to TIR1/AFBs, suppressed not only the expression of an auxin-responsive ZmSAUR2 gene, but also gravitropic curvature. Expression of ZmSAUR2 was up-regulated on the lower side and down-regulated on the upper side of the coleoptile elongation zone, corresponding to the asymmetric IAA distribution. These results indicate that the asymmetric downward streams of IAA control the differential growth rate of the cells by attenuating TIR1/AFBs-mediated auxin response genes, including ZmSAUR2, and therefore result in tropic curvature.

In this study, PEOT/PBT segmented copolymers of different compositions have been evaluated as possible scaffold materials for the tissue engineering of bone. By changing the composition of PEOT/PBT copolymers, very different mechanical and swelling behaviors are observed. Tensile strengths vary from 8 to 23 MPa and elongations at break from 500 to 1300%. Water-uptake ranges from 4 up to as high as 210%. The in vitro degradation of PEOT/PBT copolymers occurs both by hydrolysis and oxidation. In both cases degradation is more rapid for copolymers with high PEO content. PEOT/PBT scaffolds with varying porosities and pore sizes have been prepared by molding and freeze-drying techniques in combination with particulate-leaching. The most hydrophilic PEOT/PBT copolymers did not sustain goat bone marrow cell adhesion and growth. However, surface modification by gas plasma treatment showed a very much improved polymer-cell interaction for all PEOT/PBT copolymer compositions. Their mechanical properties, degradability and ability to sustain bone marrow cell growth make PEOT/PBT copolymers excellent materials for bone tissue engineering.

Amphiphilic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymers, also known as poloxamers, have broad biomembrane activities. To illustrate the nature of these activities, (1)H Overhauser dynamic nuclear polarization NMR spectroscopy was employed to sensitively detect polymer-lipid membrane interactions through the modulation of local hydration dynamics in lipid membranes. Our study shows P188, the most hydrophilic poloxamer that is a known membrane sealant, weakly adsorbs on the membrane surface, yet effectively retards membrane hydration dynamics. Contrarily, P181, the most hydrophobic poloxamer that is a known membrane permeabilizer, initially embeds at lipid headgroups and enhances intrabilayer water diffusivity. Unprecedented resolution for differentiating weak surface adsorption versus translocation of polymers to membranes is obtained by probing local water diffusivity in lipid bilayer systems. Our results illustrate that the relative hydrophilic/hydrophobic ratio of the polymer dictates its functions. These findings gleaned from local hydration dynamics are well supported by a thermodynamics study presented in the accompanying paper (Wang, J.-Y.; Marks, J. M.; Lee, K. Y. C. Biomacromolecules, 2012, DOI: 10.1021/bm300847x).

Block copolymer-based vesicles have recently garnered a great deal of interest as nanoplatforms for drug delivery and molecular imaging applications due to their unique structural properties. These nanovesicles have been shown to direct their cargo to disease sites either through enhanced permeability and retention or even more efficiently via active targeting. Here, we show that the efficacy of nanovesicle targeting can be significantly improved when prepared from polymer-lipid blends compared with block copolymer alone. Polymer-lipid hybrid nanovesicles were produced from the aqueous coassembly of the diblock copolymer, poly(ethylene oxide)-block-polybutadiene (PEO-PBD), and the phospholipid, hydrogenated soy phosphatidylcholine (HSPC). The PEG-based vesicles, 117 nm in diameter, were functionalized with either folic acid or anti-HER2/neu affibodies as targeting ligands to confer specificity for cancer cells. Our results revealed that nanovesicles prepared from polymer-lipid blends led to significant improvement in cell binding compared to nanovesicles prepared from block copolymer alone in both in vitro cell studies and murine tumor models. Therefore, it is envisioned that nanovesicles composed of polymer-lipid blends may constitute a preferred embodiment for targeted drug delivery and molecular imaging applications.

Polymer incorporation on liposomal membranes has been extensively studied as a method of enhancing the circulation time of liposomes in the bloodstream. In this study, we investigated the in vitro and in vivo characteristics of liposomes whose surface was modified using a comblike polymer comprised of a poly(methyl methacrylate) (PMMA) backbone and short poly(ethylene oxide) (PEO) side chains. Doxorubicin (DOX)-loaded liposomes incorporating with the comblike polymer were prepared and their circulation time, biodistribution and antitumor activity were evaluated in B16F10 melanoma tumor-bearing mice. The circulation half-life time in the bloodstream of the comblike polymer-incorporated liposomes (CPILs) was approximately 14- or 2-fold higher than those of the conventional or polyethyleneglycol-fixed liposomes (PEG-liposomes), respectively. Additionally, in the biodistribution assay, the accumulation of the CPILs in the tumor was higher than those of the other liposomes. Based on this result, the antitumor activities of the CPILs were higher than those of conventional liposome formulation of DOX or free DOX due to the higher passive targeting efficiency of the long-circulating CPILs to tumor. This study suggests that the incorporation of the comblike polymer on the liposomal membrane is a promising tool to further improve circulation time of liposomes in tumor-bearing mice.

Plantaricin 423, produced by Lactobacillus plantarum, and bacteriocin ST4SA produced by Enterococcus mundtii, were electrospun into nanofibers prepared from different combinations of poly(d,l-lactide) (PDLLA) and poly(ethylene oxide) (PEO) dissolved in N,N-dimethylformamide (DMF). Both peptides were released from the nanofibers with a high initial burst and retained 88% of their original antimicrobial activity at 37 °C. Nanofibers have the potential to serve as carrier matrix for bacteriocins and open a new field in developing controlled antimicrobial delivery systems for various applications.

Poly(ethylene oxide)-block-poly(styrene oxide) (PEO-b-PSO) and PEO-b-poly(butylene oxide) (PEO-b-PBO) of different chain lengths were synthesized and characterized for their self-assembling properties in water by dynamic/static light scattering, spectrofluorimetry, and transmission electron microscopy. The resulting polymeric micelles were evaluated for their ability to solubilize and protect the anticancer drug docetaxel (DCTX) from degradation. The drug release kinetics as well as the cytotoxicity of the loaded micelles were assessed in vitro. All polymers formed micelles with a highly viscous core at low critical association concentrations (<10 mg/L). Micelle morphology depended on the nature of the hydrophobic block, with PBO- and PSO-based micelles yielding monodisperse spherical and cylindrical nanosized aggregates, respectively. The maximum solubilization capacity for DCTX ranged from 0.7 to 4.2% and was the highest for PSO micelles exhibiting the longest hydrophobic segment. Despite their high affinity for DCTX, PEO-b-PSO micelles were not able to efficiently protect DCTX against hydrolysis under accelerated stability testing conditions. Only PEO-b-PBO bearing 24 BO units afforded significant protection against degradation. In vitro, DCTX was released slower from the latter micelles, but all formulations possessed a similar cytotoxic effect against PC-3 prostate cancer cells. These data suggest that PEO-b-P(SO/BO) micelles could be used as alternatives to conventional surfactants for the solubilization of taxanes.

The in vitro cytocompatibility of silicate (Laponite clay) cross-linked poly(ethylene oxide) (PEO) nanocomposite films using MC3T3-E1 mouse preosteoblast cells was investigated while cell adhesion, spreading, proliferation and mineralization were assessed as a function of film composition. By combining the advantageous characteristics of PEO polymer (hydrophilic, prevents protein and cell adhesion) with those of a synthetic and layered silicate (charged, degradable and potentially bioactive) some of the physical and chemical properties of the resulting polymer nanocomposites could be controlled. Hydration, dissolution and mechanical properties were examined and related to cell adhesion. Overall, this feasibility study demonstrates the ability of using model Laponite cross-linked PEO nanocomposites to create bioactive scaffolds.

Glass surfaces were modified by end-grafting poly(ethylene oxide) (PEO) chains having molecular weights of 526, 2000, or 9800 Da. Characterization using water contact angles, ellipsometry, and X-ray photoelectron spectroscopy confirmed the presence of the PEO brushes on the surface with estimated lengths in water of 2.8-, 7.5-, and 23.7-nm, respectively. Adhesion of two bacterial (Staphylococcus epidermidis and Pseudomonas aeruginosa) and two yeast (Candida albicans and Candida tropicalis) strains to these brushes was studied and compared to their adhesion to bare glass. For the bacterium P. aeruginosa and the yeast C. tropicalis, adhesion to the 2.8-nm brush was comparable to their adhesion on bare glass, whereas adhesion to the 7.5- and 23.7-nm brushes was greatly reduced. For S. epidermidis, adhesion was only slightly higher to the 2.8-nm brush than that to the longer brushes. Adhesion of the yeast C. albicans to the PEO brushes was lower than that to glass, but no differences in adhesion were found between the three brush lengths. After passage of an air bubble, nearly all microorganisms adhering to a brush were removed, irrespective of brush length, whereas retention of the adhering organisms on glass was much higher. No significant differences were found in adhesion nor retention between experiments conducted at 20 and those conducted at 37 degrees C.

A blend mixture of poly(epsilon-caprolactone) (PCL) and poly(ethylene oxide) (PEO) was electrospun to produce fibrous meshes that could release a protein drug in a controlled manner. Various biodegradable polymers, such as poly(l-lactic acid) (PLLA), poly(epsilon-caprolactone) (PCL), and poly(d,l-lactic-co-glycolic acid) (PLGA) were dissolved, along with PEO and lysozyme, in a mixture of chloroform and dimethylsulfoxide (DMSO). The mixture was electrospun to produce lysozyme loaded fibrous meshes. Among the polymers, the PCL/PEO blend meshes showed good morphological stability upon incubation in the buffer solution, resulting in controlled release of lysozyme over an extended period with reduced initial bursts. With varying the PCL/PEO blending ratio, the release rate of lysozyme from the corresponding meshes could be readily modulated. The lysozyme release was facilitated by increasing the amount of PEO, indicating that entrapped lysozyme was mainly released out by controlled dissolution of PEO from the blend meshes. Lysozyme released from the electrospun fibers retained sufficient catalytic activity.

The symbiosis between legume plants and rhizobia causes the development of new organs, nodules which function as an apparatus for nitrogen fixation. In this study, the roles of auxin in nodule development in Lotus japonicus have been demonstrated using molecular genetic tools and auxin inhibitors. The expression of an auxin-reporter GH3 fused to β-glucuronidase (GUS) was analyzed in L. japonicus roots, and showed a strong signal in the central cylinder of the root, whereas upon rhizobium infection, generation of GUS signal was observed at the dividing outer cortical cells during the first nodule cell divisions. When nodules were developed to maturity, strong GUS staining was detected in vascular tissues of nodules, suggesting distinct auxin involvement in the determinate nodule development. Numbers and the development of nodules were affected by auxin transport inhibitors (1-naphthylphthalamic acid, NPA and triindobenzoic acid, TIBA), and by a newly synthesized auxin antagonist, α-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA). The common phenotypical alteration by these auxin inhibitors was the inhibition in forming lenticel which is normally developed on the nodule surface from the root outer cortex. The inhibition of lenticel formation was correlated with the inhibition of nodule vascular bundle development. These results indicate that auxin is required for the normal development of determinate nodules in a multidirectional manner.

This review article focuses on thermoresponsive hydrogels consisting of poloxamers which are of high interest for biomedical application especially in drug delivery for ophthalmic, injectable, transdermal, and vaginal administration. These hydrogels remain fluid at room temperature but become more viscous gel once they are exposed to body temperature. In this way, the gelling system remains at the topical level for a long time and the drug release is controlled and prolonged. Poloxamers are synthetic triblock copolymers of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO), also commercially known as Pluronics^®, Synperonics^® or Lutrol^®. The different poloxamers cover a range of liquids, pastes, and solids, with molecular weights and ethylene oxide-propylene oxide weight ratios varying from 1100 to 14,000 and 1:9 to 8:2, respectively. Concentrated aqueous solutions of poloxamers form thermoreversible gels. In recent years this type of gel has arouse interest for tissue engineering. Finally, the use of poloxamers as biosurfactants is evaluated since they are able to form micelles in an aqueous environment above a concentration threshold known as critical micelle concentration (CMC). This property is exploited for drug delivery and different therapeutic applications.

In order to achieve long circulation time and high drug accumulation in the tumor sites via the EPR effects, anticancer drugs have to be protected by non-fouling polymers such as poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), dextran, and poly(acrylic acid) (PAA). However, the dense layer of stealth polymer also prohibits efficient uptake of anticancer drugs by target cancer cells. For cancer therapy, it is often more desirable to accomplish rapid cellular uptake after anticancer drugs arriving at the pathological site, which could on one hand maximize the therapeutic efficacy and on the other hand reduce probability of drug resistance in cells. In this review, special attention will be focused on the recent potential strategies that can enable drug-loaded polymeric nanoparticles to rapidly recognize cancer cells, leading to enhanced internalization.

Solid dispersions were prepared with the extremely poorly water soluble drug, probucol and the water soluble polymers, polyvinyl pyrrolidone (PVP), polyacrylic acid (PAA) or polyethylene oxide (PEO) and blends of these polymers. The solid dispersions were prepared either by the solvent evaporation method, or by compression moulding into films. The materials were characterised by a combination of thermal analysis and FT-Raman spectroscopy. The physical state of the drug was observed to be dependent on the carrier, thus the PVP solid dispersions contained amorphous probucol, whilst the PAA and PEO systems contained the crystalline polymorph II. The method of production was not found to greatly influence the state of the drug in the solid dispersion. The greatest extent of release into solution was observed for the binary blend of drug and PEO, and the blending of polymers was not found to have any advantageous effects in this study.

We have examined the temperature-dependent micellization of the pharmaceutically important PEO-PPO-PEO copolymer, Pluronic F127, using static light scattering and various aspects of the pyrene fluorescence spectrum (monomer intensity, excimer formation and the I1/I3 ratio). All techniques gave essentially the same value for the critical micellization temperatures (cmt) of various F127 solutions, and our results agreed with those reported in the literature. Cmt values decrease with increasing F127 concentration. We observed significant solubilization of pyrene in F127 solutions below the cmt, which was also reflected in the measured I1/I3 ratios. The thermodynamics of the micellization process were studied and gave different results at low and high F127 concentrations. In the low F127 concentration range (up to approximately 50 mg/mL), we obtain DeltaH = 312 kJ mol-1 and DeltaS = 1.14 kJ mol-1 K-1. Above 50 mg/mL we obtain DeltaH = 136 kJ mol-1 and a DeltaS = 0.54 kJ mol-1 K-1. This discontinuity in thermodynamic behavior can be due to a change in aggregation number with temperature and/or a change in the micellization process at higher concentrations. Copyright 1999 Academic Press.

Mutations in POLG gene are responsible for a wide spectrum of clinical disorders with altered mitochondrial DNA (mtDNA) integrity, including mtDNA multiple deletions and depletion. Sensory ataxic neuropathy with ophthalmoparesis (SANDO) caused by mutations in POLG gene, fulfilling the clinical triad of sensory ataxic neuropathy, dysarthria and/or dysphagia and ophthalmoparesis, has described in a few reports. Here we described five cases of adult onset autosomal recessive sensory ataxic neuropathy with ophthalmoplegia. All patients had ataxia, neuropathy, myopathy, and progressive external ophthalmoplegia (PEO). The muscle pathology revealed ragged-red and cytochrome c oxidase (COX) negative fibers in three patients. However, deficiencies in the activities of mitochondrial respiratory chain enzyme complexes were not detected in any of the patients' muscle samples. Multiple deletions of mtDNA were detected in blood and muscle specimens but mtDNA depletion was not found. Due to these diagnostic difficulties, POLG-related syndromes are definitively diagnosed based on the presence of deleterious mutations in the POLG gene.

The pH-induced release of hydrophilic dyes from poly(2-vinylpyridine-b-ethylene oxide) (P2VP-PEO) block copolymer vesicles is investigated. The structure of the vesicles is characterized using small-angle neutron scattering (SANS) and cryo-electron microscopy (cryo-TEM). A decrease of the pH below 5 leads to protonation and dissolution of the poly-2-vinylpyridine blocks which induces rupture and dissolution of the vesicle membrane. Details of the rupture, dissolution, and release process are studied by fluorescence video microscopy, gel electrophoresis, and high-performance ultrafiltration.

Superparamagnetic iron oxide particles (SPIO) of maghemite were prepared in aqueous solution and subsequently stabilized with polymers in two layer-by-layer deposition steps. The first layer around the maghemite core is formed by poly(ethylene imine) (PEI), and the second one is formed by poly(ethylene oxide)-block-poly(glutamic acid) (PEO-PGA). The hydrodynamic diameter of the particles increases stepwise from D(h) = 25 nm (parent) via 35 nm (PEI) to 46 nm (PEI plus PEO-PGA) due to stabilization. This is accompanied by a switching of their zeta-potentials from moderately positive (+28 mV) to highly positive (+50 mV) and finally slightly negative (-3 mV). By contrast, the polydispersity indexes of the particles remain constant (ca. 0.15). Mössbauer spectroscopy revealed that the iron oxide, which forms the core of the particles, is only present as Fe(III) in the form of superparamagnetic maghemite nanocrystals. The magnetic domains and the maghemite crystallites were found to be identical with a size of 12.0 +/- 0.5 nm. The coated maghemite nanoparticles were tested to be stable in water and in physiological salt solution for longer than 6 months. In contrast to novel methods for magnetic nanoparticle production, where organic solvents are necessary, the procedure proposed here can dispense with organic solvents. Magnetic resonance imaging (MRI) experiments on living rats indicate that the nanoparticles are useful as an MRI contrast agent.

BACKGROUND

This study evaluates the eye drop delivery of genes with cornea-specific promoters, i.e., keratin 12 (K12) and keratocan (Kera3.2) promoters, by non-ionic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles (PM) to mouse and rabbit eyes, and investigates the underlying mechanisms.

METHODS

Three PM-formulated plasmids (pCMV-Lac Z, pK12-Lac Z and pKera3.2-Lac Z) containing the Lac Z gene for beta-galactosidase (beta-Gal) whose expression was driven by the promoter of either the cytomegalovirus early gene, the keratin 12 gene or the keratocan gene, were characterized by critical micelle concentration (CMC), dynamic light scattering (DLS), and atomic force microscopy (AFM). Transgene expression in ocular tissue after gene delivery was analyzed by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) color staining, 1,2-dioxetane beta-Gal enzymatic activity measurement, and real-time polymerase chain reaction (PCR) analysis. The delivery mechanisms of plasmid-PM on mouse and rabbit corneas were evaluated by EDTA and RGD (arginine-glycine-aspartic acid) peptide.

RESULTS

The sizes of the three plasmid-PM complexes were around 150-200 nm with unimodal distribution. Enhanced stability was found for three plasmid-PM formulations after DNase I treatment. After six doses of eye drop delivery of pK12-Lac Z-PM three times a day, beta-Gal activity was significantly increased in both mouse and rabbit corneas. Stroma-specific Lac Z expression was only found in pKera3.2-Lac Z-PM-treated animals with pretreatment by 5 mM EDTA, an opener of junctions. Lac Z gene expression in both pK12-Lac Z-PM and pKera3.2-Lac Z-PM delivery groups was decreased by RGD peptide pretreatment.

CONCLUSIONS

Cornea epithelium- and stroma-specific gene expression could be achieved using cornea-specific promoters of keratin 12 and keratocan genes, and the gene was delivered with PM formulation through non-invasive, eye drop in mice and rabbits. The transfection mechanism of plasmid-PM may involve endocytosis and particle size dependent paracellular transport.