Precise Synthesis and Thin Film Self-Assembly of PLLA-b-PS Bottlebrush Block Copolymers.

The ability of bottlebrush block copolymers (BBCPs) to self-assemble into ordered large periodic structures could greatly expand the scope of photonic and membrane technologies. In this paper, we describe a two-step synthesis of poly(l-lactide)-b-polystyrene (PLLA-b-PS) BBCPs and their rapid thin-film self-assembly. PLLA chains were grown from exo-5-norbornene-2-methanol via ring-opening polymerization (ROP) of l-lactide to produce norbornene-terminated PLLA. Norbonene-terminated PS was prepared using anionic polymerization followed by a termination reaction with exo-5-norbornene-2-carbonyl chloride. PLLA-b-PS BBCPs were prepared from these two norbornenyl macromonomers by a one-pot sequential ring opening metathesis polymerization (ROMP). PLLA-b-PS BBCPs thin-films exhibited cylindrical and lamellar morphologies depending on the relative block volume fractions, with domain sizes of 46-58 nm and periodicities of 70-102 nm. Additionally, nanoporous templates were produced by the selective etching of PLLA blocks from ordered structures. The findings described in this work provide further insight into the controlled synthesis of BBCPs leading to various possible morphologies for applications requiring large periodicities. Moreover, the rapid thin film patterning strategy demonstrated (>5 min) highlights the advantages of using PLLA-b-PS BBCP materials beyond their linear BCP analogues in terms of both dimensions achievable and reduced processing time.

Efficacy of end-only-functionalized oligo(arylene-ethynylene)s in killing bacterial biofilms.

Cationic end-only-functionalized oligo(arylene-ethynylene)s (EO-OPEs) have recently been found to be broad-spectrum and effective antimicrobial agents because of their unique structure and optical properties. In this study, we investigated their potential use for preventing and reducing Escherichia coli (E. coli) biofilms. The Calgary biofilm device (CBD) was used to form bacterial biofilms of E. coli; in these studies, the minimum inhibitory concentration (MIC) and the minimum biofilm eradication concentration (MBEC) were determined. E. coli biofilms uniformly grow on pegs of the CBD device lid. The MIC values determined for EO-OPEs are comparable to those found for standard antibiotics such as kanamycin (MIC = 11.2 µg/mL). About 10-30 times the concentration of EO-OPEs was required to eradicate E. coli biofilms and prevent regrowth in the dark. Near-UV irradiation of EO-OPEs enhanced their efficacy in killing biofilms.

pH-dependent optical properties of a poly(phenylene ethynylene) conjugated polyampholyte.

A poly(phenylene ethynylene) conjugated polymer (PPE-NMe(3)(+)-COO(-)) containing tetraalkylammonium groups and carboxylate groups has been synthesized by Sonogashira coupling. Due to the presence of the strong cationic and weak anionic pendant units, the polymer undergoes a pH-induced transition from cationic polyelectrolyte to polyampholyte due to deprotonation of the carboxylic acid units in basic solution. Studies of the pH dependence of the polymers' optical properties reveal changes in absorption oscillator strength and fluorescence quantum efficiency that are triggered by the transition from cationic polyelectrolyte to polyampholyte nature. Stern-Volmer fluorescence quenching of PPE-NMe(3)(+)-COO(-) with a negatively charged quencher 1,4,5,8-naphthalenediimide-N,N-bis(methylsulfonate) (NDS) shows that the polymer fluorescence quenching is amplified at low pH where the polymer is a polycation, whereas the quenching efficiency is considerably less at high pH.

Antibacterial activity of conjugated polyelectrolytes with variable chain lengths.

Cationic poly(phenylene ethynylene)- (PPE-) based conjugated polyelectrolytes (CPEs) with six different chain lengths ranging in degree of polymerization from ∼7 to ∼49 were synthesized from organic-soluble precursor polymers. The molecular weight of the precursor polymers was controlled by the amount of a monofunctional "end-capping" agent added to the polymerization reaction. Cationic CPEs were prepared by quaternization of amine groups to tetraalkylammonium groups. Their structure-property relationships were investigated by observing their photophysical properties and antibacterial activity. The polymers were found to exhibit a chain-length dependence in their photophysical properties. It has also been observed that the polymers exhibit effective antibacterial activity against both Gram-positive and Gram-negative bacteria under UV irradiation, whereas they show little antibacterial activity in the dark. An effect of chain length on the light-activated antibacterial activity was also found: The shortest polymer (n=7) exhibited the most effective antibacterial activity against both Gram-positive and Gram-negative bacteria.

Effect of polymer chain length on membrane perturbation activity of cationic phenylene ethynylene oligomers and polymers.

The interactions of poly(phenylene ethynylene)- (PPE-) based cationic conjugated polyelectrolytes (CPEs) and oligo(phenylene ethynylene)s (OPEs) with different model lipid membrane systems were investigated to gain insight into the relationship between molecular structure and membrane perturbation ability. The CPE and OPE compounds exhibit broad-spectrum antimicrobial activity, and cell walls and membranes are believed to be their main targets. To better understand how the size, in terms of the number of repeat units, of the CPEs and OPEs affects their membrane disruption activities, a series of PPE-based CPEs and OPEs were synthesized and studied. A number of photophysical techniques were used to investigate the interactions of CPEs and OPEs with model membranes, including unilamellar vesicles and lipid monolayers at the air/water interface. CPE- or OPE-induced dye leakage from vesicles reveals that the CPEs and OPEs selectively perturb model bacterial membranes and that their membrane perturbation abilities are highly dependent on molecular size. Consistent with dye-leakage assay results, the CPEs and OPEs also exhibit chain-length-dependent ability to insert into 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) monolayers. Our results suggest that, for PPE-based CPE and OPE antimicrobials, chain length can be tuned to optimize their membrane perturbation ability.

Intercalation-FRET biosensor with a helical conjugated polyelectrolyte.

A biotin-tetramethylrhodamine (biotin-TMR) quencher-ligand interacts with a (phenylene-ethynylene) based helical conjugated polyelectrolyte (poly-1) via intercalation of the TMR unit into the helix. The interaction is signaled by efficient fluorescence resonance energy transfer (FRET) from the polymer to the TMR chromophore. Avidin addition to the poly-1/biotin-TMR intercalation complex does not interrupt FRET, instead resulting in the formation of avidin-biotin "cross-links". Mixing of biotin-TMR with avidin prior to addition of the polymer efficiently disrupts the FRET signal, giving rise to a sensor with a detection limit of 100 pM for avidin. Study of the FRET response as a function of biotin-TMR and avidin concentration affords insight into the interaction of the protein with the poly-1/biotin-TMR intercalation complex.

Membrane perturbation activity of cationic phenylene ethynylene oligomers and polymers: selectivity against model bacterial and mammalian membranes.

Poly(phenylene ethyneylene) (PPE)-based cationic conjugated polyelectrolytes (CPEs) and cationic phenylene ethynylene oligomers (OPEs) exhibit broad-spectrum antimicrobial activity, and their main target is believed to be the cell membrane. To understand better how these antimicrobial molecules interact with membranes, a series of PPE-based CPEs and OPEs with different side chains were studied. Large unilamellar vesicles with lipid compositions mimicking those of mammalian or bacterial membranes were used as model membranes. Among the CPEs and OPEs tested, the anionic CPE, PPE-SO(3)(2-) and the smallest cationic OPE-1 are inactive against all vesicles. Other cationic CPEs and OPEs show significant membrane perturbation ability against bacterial membrane mimics but are inactive against a mammalian cell membrane mimic with the exception of PPE-DABCO and two end-only-functionalized OPEs, which also disrupted a mammalian cell membrane mimic. The results suggest that the phospholipid composition of vesicles dominates the interaction of CPE and OPE with lipid membranes.

Insight into the mechanism of antimicrobial poly(phenylene ethynylene) polyelectrolytes: interactions with phosphatidylglycerol lipid membranes.

The interactions of antimicrobial poly(phenylene ethynylene) (PPE)-based cationic conjugated polyelectrolytes (CPEs) with lipid membranes were investigated to gain insight into the mechanism of their biocidal activity. Three model membrane systems comprising negatively charged phosphatidylglycerol (PG) lipids were used to mimic the bacterial cell membrane, including unilamellar lipid vesicles in aqueous solution, lipid bilayer coated silica microspheres, and lipid monolayers at the air-water interface. Two PPE CPEs, one containing a thiophene moiety on the PPE repeat unit and the second containing a diazabicyclooctane (DABCO) moiety on the pendant side chain, were chosen, since the former exhibits distinct dark biocidal activity and the latter shows strong light-activated antimicrobial activity but little dark biocidal activity. The interactions of these two PPE polymers with lipid membranes were characterized in detail by CPE fluorescence spectral changes, fluorescence resonance energy transfer (FRET), fluorescence quenching, monolayer insertion, and dynamic light scattering assays. Both PPE polymers exhibit affinity for the anionic lipid membrane systems. Their concomitant association and insertion into the membrane leads to conformational changes of the PPE polymer from an aggregated state to a more extended state, as evidenced by the polymer's enhanced fluorescence and FRET between the polymer and rhodamine incorporated in the lipid membrane. In comparison, the thiophene polymer exhibits stronger interactions with PG lipid membranes than the DABCO-containing polymer. The former induces a larger fluorescence enhancement, shows faster transfer across the lipid membrane, and inserts more readily and to a higher extent into lipid monolayers. The observed differences between the two PPE polymers in their interactions with the lipid membrane may stem from their structural differences, as the DABCO-containing polymer has a much bulkier and larger pendant group on its side chain. The higher degree of membrane interaction and insertion, and subsequent membrane disorganization, of the thiophene polymer may account for its dark biocidal activity.

Conjugated polyelectrolyte supported bead based assays for phospholipase A2 activity.

A fluorescence based assay for human serum-derived phospholipase activity has been developed in which cationic conjugated polyelectrolytes are supported on silica microspheres. The polymer-coated beads are overcoated with an anionic phospholipid (1,2-dimyristoyl-sn-glycero-3-[phospho- rac-(1-glycerol)) (DMPG) to provide "lipobeads" that serve as a sensor for PLA2. The lipid serves a dual role as a substrate for PLA2 and an agent to attenuate quenching of the polymer fluorescence by the external electron transfer quencher 9,10-anthraquinone-2,6-disulfonic acid (AQS). In this case quenching of the polymer fluorescence by AQS increases as the PLA2 digests the lipid. The lipid can also be used itself as a quencher and substrate by employing a small amount of energy transfer quencher substituted lipid in the DMPG. In this case the fluorescence of the polymer is quenched when the lipid layer is intact; as the enzyme digests the lipid, the fluorescence of the polymer is restored. The sensing of PLA2 activity has been studied both by monitoring fluorescence changes in a multiwell plate reader and by flow cytometry. The assay exhibits good sensitivity with EC50 values in the nanomolar range.

Hierarchical honeycomb-structured films by directed self-assembly in "breath figure" templating of ionizable "clicked" PH3T-b-PMMA diblock copolymers: an ionic group/counter-ion effect on porous polymer film morphology.

The self-assembly of 1,2,3-triazole and ionic 1,2,3-triazolium "clicked" poly(3-hexylthiophene)-b-poly(methylmethacrylate) (P3HT-b-PMMA) rod-coil diblock copolymers was used to fabricate honeycomb-patterned porous films via "breath figure" templating. The surface and inner morphologies of the honeycomb films can be both controlled by either ionizing the 1,2,3-triazole linker or changing the counter-ion nature.

Gender differences in the corpus callosum of neonates.

The gender difference of corpus callosum is a long standing controversy. Some investigators have reported that adult females have a more bulbous splenium and larger area relative to brain size, but others failed to replicate this. Few studies have reported on sexual dimorphism during development. Midsagittal ultrasonographs were obtained through the anterior fontanelle of healthy Korean neonates (100 males, 100 females) and were analyzed with NIH Image. The total area of corpus callosum and sub-regions of corpus callosum showed no gender differences. However, the male corpus callosum had significantly more height than that of the female whereas there was no difference in width of the corpus callosum. The estimated average thickness of corpus callosum was greater in the female splenium.

Photophysics and light-activated biocidal activity of visible-light-absorbing conjugated oligomers.

The photophysical properties of three cationic π-conjugated oligomers were correlated with their visible light activated biocidal activity vs S. aureus. The oligomers contain three arylene units (terthiophene, 4a; thiophene-benzotriazole-thiophene, 4b; thiophene-benzothiadiazole-thiophene, 4c) capped on each end by cationic -(CH2)3NMe3(+) groups. The oligomers absorb in the visible region due to their donor-acceptor-donor electronic structure. Oligomers 4a and 4b have high intersystem crossing and singlet oxygen sensitization efficiency, but 4c has a very low intersystem crossing efficiency and it does not sensitize singlet oxygen. The biocidal activity of the oligomers under visible light varies in the order 4a > 4b ≈ 4c.

Light and dark-activated biocidal activity of conjugated polyelectrolytes.

This Spotlight on Applications provides an overview of a research program that has focused on the development and mechanistic study of cationic conjugated polyelectrolytes (CPEs) that function as light- and dark-active biocidal agents. Investigation has centered on poly-(phenylene ethynylene) (PPE) type conjugated polymers that are functionalized with cationic quaternary ammonium solubilizing groups. These polymers are found to interact strongly with Gram-positive and Gram-negative bacteria, and upon illumination with near-UV and visible light act to rapidly kill the bacteria. Mechanistic studies suggest that the cationic PPE-type polymers efficiently sensitize singlet oxygen ((1)O(2)), and this cytotoxic agent is responsible for initiating the sequence of events that lead to light-activated bacterial killing. Specific CPEs also exhibit dark-active antimicrobial activity, and this is believed to arise due to interactions between the cationic/lipophilic polymers and the negatively charged outer membrane characteristic of Gram-negative bacteria. Specific results are shown where a cationic CPE with a degree of polymerization of 49 exhibits pronounced light-activated killing of E. coli when present in the cell suspension at a concentration of 1 µg mL(-1).

Conjugated-polyelectrolyte-grafted cotton fibers act as "micro flypaper" for the removal and destruction of bacteria.

We demonstrate herein a method for chemically modifying cotton fibers and cotton-containing fabric with a light-activated, cationic phenylene-ethynylene (PPE-DABCO) conjugated polyelectrolyte biocide. When challenged with Pseudomonas aeruginosa and Bacillus atropheaus vegetative cells from liquid suspension, light-activated PPE-DABCO effects 1.2 and 8 log, respectively, losses in viability of the exposed bacteria. These results suggest that conjugated polyelectrolytes retain their activity when grafted to fabrics, showing promise for use in settings where antimicrobial textiles are needed.

Light and dark biocidal activity of cationic poly(arylene ethynylene) conjugated polyelectrolytes.

In this paper we report a study of cationic poly(arylene ethynylene) conjugated polyelectrolytes. The objective of the study was to compare the behavior of a polymer where a thiophene has replaced a phenyl ring in poly(phenylene ethynylene) polycations (PPE) previously investigated. Properties of solution phase and physisorbed suspensions of the polymer on microspheres were investigated. The photophysical properties of the polymer are evaluated and used to understand the striking differences in biocidal activity compared to the PPE polymers previously examined. The principal findings are that the thiophene polymer has remarkable dark biocidal activity against Pseudomonas aeruginosa strain PAO1 but very little light-activated activity. The low light-activated biocidal activity of the thiophene polymer is attributed to a highly aggregated state of the polymer in aqueous solutions and on microspheres as a physisorbed coating. This results in low triplet yields and a very poor sensitization of singlet oxygen and other reactive oxygen intermediates. The highly effective dark biocidal activity of the thiophene-containing polymers is attributed to its high lipophilicity and the presence of accessible quaternary ammonium groups. The difference in behavior among the polymers compared provides insights into the mechanism of the dark process and indicates that aggregation of polymer can reduce light activated biocidal activity by suppressing singlet oxygen generation.

Light-induced biocidal action of conjugated polyelectrolytes supported on colloids.

A series of water soluble, cationic conjugated polyelectrolytes (CPEs) with backbones based on a poly(phenylene ethynylene) repeat unit structure and tetraakylammonium side groups exhibit a profound light-induced biocidal effect. The present study examines the biocidal activity of the CPEs, correlating this activity with the photophysical properties of the polymers. The photophysical properties of the CPEs are studied in solution, and the results demonstrate that direct excitation produces a triplet excited-state in moderate yield, and the triplet is shown to be effective at sensitizing the production of singlet oxygen. Using the polymers in a format where they are physisorbed or covalently grafted to the surface of colloidal silica particles (5 and 30 microm diameter), we demonstrate that they exhibit light-activated biocidal activity, effectively killing Cobetia marina and Pseudomonas aeruginosa. The light-induced biocidal activity is also correlated with a requirement for oxygen suggesting that interfacial generation of singlet oxygen is the crucial step in the light-induced biocidal activity.

Parental artistic drawings of the fetus before and after 3-/4-dimensional ultrasonography.

OBJECTIVE: The purpose of this study was to assess the impact of 3-/4-dimensional ultrasonography (3D/4DUS) on parental impressions of their fetus using preexamination and postexamination parental drawings. METHODS: One hundred one parents (32 male and 69 female) were asked to draw pictures of their fetus immediately before and after undergoing 3D/4DUS. A drawing assessment instrument was used to evaluate the fetal anatomy by 4 reviewers. Overall descriptive quality and a comparison of before and after images were assessed. RESULTS: Reviewers judged the drawings as significantly different in 23% to 56% of cases, slightly different in 41% to 64%, and having no significant difference in 2% to 22%; some difference was identified in at least 78%. Reviewers familiar with ultrasonography were able to correctly predict which picture was drawn first in 78%. Differences identified were related to extremity positioning, personalized uterine environment, and artistic nature. No significant difference in the overall total scoring for anatomic differentiation before and after 3D/4DUS was found. A trend in drawing less anatomic structures after 3D/4DUS was discovered. Significantly fewer people drew hair after 3D/4DUS (P<.01) even when they had drawn hair before the 3D/4DUS. Similar significant differences were seen for the lower extremities (P<.01), with less detail being presented after the 3D/4DUS. CONCLUSIONS: Differences were seen between drawings made before and after 3D/4DUS by parents. Drawings after 3D/4DUS emphasized structures seen during the 3D/4DUS examination and were more reality based, whereas drawings before 3D/4DUS were more iconographic and based on preconceived anatomic knowledge.

Preexamination and postexamination assessment of parental-fetal bonding in patients undergoing 3-/4-dimensional obstetric ultrasonography.

OBJECTIVE: The purpose of this study was to determine whether there is a change in parental bonding and couples' attitudes toward their fetus after undergoing 3-/4-dimensional ultrasonography (3D/4DUS). METHODS: Sixty-five fathers and 124 mothers were asked to fill out a maternal-fetal attachment questionnaire relating to how they felt about their fetus before and after 3D/4DUS and to mark on a line indicating their feelings about the ultrasonography experience. In addition, 135 parents filled out a positive feelings questionnaire consisting of 5 sections assessing their feelings about the fetus. The 3D/4DUS examination included rendering of the fetal face, limbs, and thorax. RESULTS: One hundred forty-two patients filled out all questions and were analyzed for the total attachment score. The difference of the total score for the maternal-fetal attachment questionnaire before and after 3D/4DUS had a z value of 5.6 for all patients and was statistically significant (P < .0001). In analyzing each question, 5 were found to have a statistically significantly different score for women, but only 2 were found so for men. The scores for the line, before and after 3D/4DUS, showed a significant difference for men but not women. The women studied did not show a change using this instrument because their median response was at the maximum measurement before their sonograms. The positive feelings questionnaire showed a statistically significant change for women in all sections but for men in only 2. CONCLUSIONS: Parents have a change in attitude regarding their fetus after undergoing 3D/4DUS. Mothers showed an increase in bonding to their fetus after 3D/4DUS in more categories than fathers.

Isolated echogenic foci in the left upper quadrant of the fetal abdomen: are they significant?

OBJECTIVES: To evaluate fetuses with echogenic foci in the left upper quadrant of the abdomen by sonographic examination. METHODS: Between November 2000 and October 2001, we prospectively assessed fetuses with echogenic foci in the abdominal left upper quadrant on routine prenatal sonography. The location, number, and size of the foci were analyzed, and in utero and postpartum follow-up scans were performed. A pediatrician evaluated the perinatal outcome. RESULTS: Twenty-six fetuses had 35 echogenic foci in the left upper quadrant of the abdomen at gestational ages of 20 to 37 weeks. The locations of the foci along the stomach, spleen, and the left lobe of the liver were found on sonography and measured 2 to 5 mm. Of the 16 fetuses who had follow-up scans in utero, 7 had disappearance of the foci. Postpartum follow-up scans were performed in 12 cases. Among them, 9 neonates had no lesions, and 3 had echogenic foci of a size and appearance similar to those seen prenatally in the left lobe of the liver. Only 1 of 4 patients who had TORCH (toxoplasmosis, other, rubella, cytomegalovirus, and herpes simplex) titers evaluated had elevated immunoglobulin G levels; no patient had any sign of infection after birth. All fetuses had a normal postnatal outcome. CONCLUSIONS: Many echogenic foci in the left upper quadrant of the fetal abdomen will disappear, and the outcome is promising even when the lesion persists after birth.