Uniform conductive composite membranes were prepared using a phase inversion method by blending carboxyl-functionalized multi-walled carbon nanotubes (CNTs) with a polysulfone polymer. At 6 % of the embedded CNTs, the membrane pore size measured by transmission electron microscopy (TEM) was approximately 50 nm. Electric current in the presence of the composite membranes markedly inactivated the model pathogenic bacteria Escherichia coli and Staphylococcus aureus, with the extent of bacterial inactivation rising when the current was increased. Over 99.999 % inactivation of both bacteria was observed in deionized water after 40 min at 5 mA direct current (DC); importantly, no appreciable inactivation occurred in the absence of either the electric field or the CNTs within the membranes under otherwise the same conditions. A much lower, although still pronounced, inactivation was seen with alternating current (AC) in a 25 mM NaCl aqueous solution.

Several diverse proteins are found to readily dissolve in neat low-transition-temperature mixtures (LTTMs). They undergo no irreversible denaturation in such unusual solvents, and the resistance of hen egg-white lysozyme against thermoinactivation in LTTMs is greater than in aqueous solution at extreme pHs. Separately, the water-sensitive drug aspirin is found to form concentrated transparent LTTMs, where it is some 10-fold more stable against cleavage than in water.

The currently used multistep chemical synthesis for making surfaces antimicrobial by attaching to them hydrophobic polycations is replaced herein by an aerosol-assisted plasma deposition procedure. To this end, N,N-hexyl,methyl-PEI (HMPEI) is directly plasma-coated onto a glass surface. The resultant immobilized HMPEI coating has been thoroughly characterized and shown to be robust, bactericidal against Escherichia coli, and virucidal against human influenza virus.

Previously, polymer-attached zanamivir had been found to inhibit influenza A viruses in vitro far better than did small-molecule zanamivir (1) itself. The aim of this study was to identify in vitro—using the plaque reduction assay—a highly potent 1-polymer conjugate, and subsequently test its antiviral efficacy in vivo.By examining the structure-activity relationship of 1-polymer conjugates in the plaque assay, we have determined that the most potent inhibitor against several representative influenza virus strains has a neutral high-molecular-weight backbone and a short alkyl linker. We have examined this optimal polymeric inhibitor for efficacy and immunogenicity in the mouse and ferret models of infection.1 attached to poly-L-glutamine is an effective therapeutic for established influenza infection in ferrets, reducing viral titers up to 30-fold for 6 days. There is also up to a 190-fold reduction in viral load when the drug is used as a combined prophylactic/therapeutic in mice. Additionally, we see no evidence that the drug conjugate stimulates an immune response in mice upon repeat administration.1 attached to a neutral high-molecular-weight backbone through a short alkyl linker drastically reduced both in vitro and in vivo titers compared to those observed with 1 itself. Thus, further development of this polymeric zanamivir for the mitigation of influenza infection seems warranted.

To explore surface-immobilized and suspended modalities of the hydrophobic polycation N,N-dodecyl,methyl-polyethylenimine (DMPEI) for the ability to reduce viral infectivity in aqueous solutions containing herpes simplex viruses (HSVs) 1 and 2.Surface-immobilized (coated onto surfaces) and suspended DMPEI were incubated with aqueous solutions containing HSV-1 or -2 to measure the antiviral effect of the hydrophobic polycation’s formulations on HSVs.DMPEI coated on either polyethylene slides or male latex condoms dramatically decreases infectivity in solutions containing HSV-1 or -2. Moreover, DMPEI suspended in aqueous solution markedly reduces the infectious titer of these HSVs.Our results suggest potential uses of DMPEI for both prophylaxis (in the form of coated condoms) and treatment (as a topical suspension) for HSV infections.

The aim of this study was to markedly lower the viscosities of highly concentrated protein, in particular antibody, formulations. An effective approach elaborated herein for γ-globulin and a monoclonal antibody is to replace aqueous solutions with equimolar suspensions in neat organic solvents.Viscosities of aqueous solutions and non-aqueous suspensions of the model protein bovine γ-globulin and a murine monoclonal antibody were examined under a variety of experimental conditions. In addition, protein particle sizes were measured using dynamic light scattering and light microscopy.Concentrated suspensions of amorphous γ-globulin powders (up to 300 mg/mL, composed of multi-micron-sized particles) in absolute ethanol and a number of other organic solvents were found to have viscosities up to 38 times lower than the corresponding aqueous solutions. Monoclonal antibody follows the same general trend. Additionally, the higher the protein concentration and lower the temperature, the greater the viscosity benefit of a suspension over a solution.The viscosities of concentrated γ-globulin and monoclonal antibody suspensions in organic solvents are drastically reduced compared to the corresponding aqueous solutions; the magnitude of this reduction depends on the solvent, particularly its hydrogen-bonding properties.

Heparin covalently attached to a water-insoluble resin suspended in HIV-infected aqueous buffer or whole blood captures the virus; subsequent physical separation of the immobilized heparin reduced the viral titers by over 80 and 50%, respectively. The detoxification concept has been validated by both circulating an HIV-1 solution through a column packed with the heparin–sepharose beads and successively mixing an HIV-1 solution with fresh beads.

Covalently conjugating multiple copies of the drug zanamivir (ZA; the active ingredient in Relenza) via a flexible linker to poly-l-glutamine (PGN) enhances the anti-influenza virus activity by orders of magnitude. In this study, we investigated the mechanisms of this phenomenon. Like ZA itself, the PGN-attached drug (PGN-ZA) binds specifically to viral neuraminidase and inhibits both its enzymatic activity and the release of newly synthesized virions from infected cells. Unlike monomeric ZA, however, PGN-ZA also synergistically inhibits early stages of influenza virus infection, thus contributing to the markedly increased antiviral potency. This inhibition is not caused by a direct virucidal effect, aggregation of viruses, or inhibition of viral attachment to target cells and the subsequent endocytosis; rather, it is a result of interference with intracellular trafficking of the endocytosed viruses and the subsequent virus-endosome fusion. These findings both rationalize the great anti-influenza potency of PGN-ZA and reveal that attaching ZA to a polymeric chain confers a unique mechanism of antiviral action potentially useful for minimizing drug resistance.

To discover, elucidate the structure-activity relationship (SAR), and explore the mechanism of action of excipients able to drastically lower the viscosities of concentrated aqueous solutions of humanized monoclonal antibodies (MAbs).Salts prepared from hydrophobic cations and anions were dissolved into humanized MAbs solutions. Viscosities of the resulting solutions were measured as a function of the nature and concentration of the salts and MAbs.Even at moderate concentrations, some of the salts prepared herein were found to reduce over 10-fold the viscosities of concentrated aqueous solutions of several MAbs at room temperature.To be potent viscosity-lowering excipients, the ionic constituents of the salts must be hydrophobic, bulky, and aliphatic. A mechanistic hypothesis explaining the observed salt effects on MAb solutions’ viscosities was proposed and verified.

N,N-Dodecyl,methyl-polyethylenimine coatings applied to solid surfaces have been shown by us to disinfect aqueous solutions of influenza viruses. Herein we elucidate the mechanism of this phenomenon. Infectivity-, protein-, RNA-, and scanning electron microscopy-based experiments reveal that, upon contact with the hydrophobic polycationic coating, influenza viruses (including pathogenic human and avian, both wild-type and drug-resistant, strains) irreversibly adhere to it, followed by structural damage and inactivation; subsequently, viral RNA is released into solution, while proteins remain adsorbed.

A methodology is developed and validated whereby a cotton fabric is impregnated with a photosensitive hydrophobic N-alkyl-polyethylenimine, followed by its covalent immobilization triggered by ultraviolet light. The resultant fabric efficiently kills on contact waterborne pathogenic bacteria E. coli and S. aureus.

New antiviral agents were prepared by attaching derivatives of sialic acid (1) and of the drug zanamivir (2) to poly(isobutylene-alt-maleic anhydride) (poly-(1 + 2)) or by mixing poly-1 and poly-2, followed by assaying them against wild-type and drug-resistant influenza A Wuhan viruses.Individually or together, 1 and 2 were covalently bonded to the polymer. The antiviral potencies of the resultant poly-1, poly-2, poly-(1 + 2), and poly-1 + poly-2, as well as 1 and 2, were assessed using plaque reduction assay.Attaching 1 to the polymer improved at best millimolar IC50 values over three orders of magnitude. While 2 exhibited micromolar IC50 values, poly-2 was >100-fold even more potent. The IC50 of poly-(1 + 2) against the wild-type strain was >300-fold and ∼17-fold better than of poly-1 and poly-2, respectively. In contrast, the potency of poly-(1 + 2) vs. poly-2 against the mutant strain merely doubled. The mixture of poly-1 + poly-2 inhibited both viral strains similarly to poly-2.The bifunctional poly-(1 + 2) acts synergistically against the wild-type influenza virus, but not against its drug-resistant mutant, as compared to a physical mixture of the monofunctional poly-1 and poly-2.

The effect of all possible mutations at position 178 on the enantioselectivity of yeast surface-bound horseradish peroxidase (HRP) toward chiral phenols has been investigated. In contrast to their wild-type predecessor, most HRP mutants are enantioselective, with the Arg178Glu variant exhibiting the greatest, 25-fold, (S)/(R) preference. Using kinetic analysis of enzymatic oxidation of various substrate analogues and molecular modeling of enzyme−substrate complexes, this enantioselectivity enhancement is attributed to changes in the transition state energy due to electrostatic repulsion between the carboxylates of the enzyme’s Glu178 and the substrate’s (R)-enantiomer.

We previously discovered that coating solid surfaces with long-chained linear N-dodecyl,N-methyl-polyethylenimine makes them bactericidal and virucidal. In the present study, focusing on the use of this microbicidal paint to kill airborne Escherichia coli and Staphylococcus aureus, we have systematically investigated the dependence of this effect on the concentration and mode of application of the hydrophobic polycation, the number of coats, the nature of the solvent, and the presence of a dye in such paint. In addition, the latter’s ability to be regenerated after use, stability upon repeated washings, and mammalian toxicity has been evaluated.

Glass slides painted with the hydrophobic long-chained polycation N,N-dodecyl,methyl-polyethylenimine (N,N-dodecyl,methyl-PEI) are highly lethal to waterborne influenza A viruses, including not only wild-type human and avian strains but also their neuraminidase mutants resistant to currently used anti-influenza drugs.

A highly efficient selection method for enhanced enzyme enantioselectivity based on yeast surface display and fluorescence-activated cell sorting (FACS) is developed and validated. Its application to horseradish peroxidase has resulted in enzyme variants up to 2 orders of magnitude selective toward either substrate enantiomer at will. These marked improvements in enantioselectivity are demonstrated for the surface-bound and soluble enzymes and rationalized by computational docking studies.

To investigate the moisture-induced aggregation (i.e., a loss of solubility in water) of DNA in a solid state and to develop rational strategies for its prevention.Lyophilized calf thymus DNA was exposed to relative humidity (RH) levels from 11% to 96% at 55°C. Following a 24-h incubation under these stressed conditions, the solubility of DNA in different aqueous solutions and the water uptake of DNA were determined. The effects of solution pH and NaCl concentration and the presence of excipients (dextran and sucrose) on the subsequent moisture-induced aggregation of DNA were examined. The extent of this aggregation was compared with that of a supercoiled plasmid DNA.Upon a 24-h incubation at 55°C, calf thymus DNA underwent a major moisture-induced aggregation reaching a maximum at a 60% RH; in contrast, the single-stranded DNA exhibited the maximal aggregation at a 96% RH. Moisture uptake and aqueous solubility studies revealed that the aggregation was primarily due to formation of inter-strand hydrogen bonds. Aggregation of DNA also proceeded at 37°C, albeit at a slower rate. Solution pH and NaCl concentration affected DNA aggregation only at higher RH levels. This aggregation was markedly reduced by co-lyophilization with dextran or sucrose (but not with PEG). The aggregation pattern of a supercoiled plasmid DNA was similar to that of its linear calf thymus counterpart.The moisture-induced aggregation of lyophilized DNA is caused mainly by non-covalent cross-links between disordered, single-stranded regions of DNA. At high RH levels, renaturation and aggregation of DNA compete with each other. The aggregation is minimized at low RH levels, at optimal solution pH and salt concentration prior to lyophilization, and by co-lyophilizing with excipients capable of forming multiple hydrogen bonds, e.g., dextran and sucrose.

Low efficiency and toxicity are two major drawbacks of current non-viral gene delivery vectors. Since DNA delivery to mammalian cells is a multi-step process, generating and searching combinatorial libraries of vectors employing high-throughput synthesis and screening methods is an attractive strategy for the development of new improved vectors because it increases the chance of identifying the most overall optimized vectors.Based on the rationale that increasing the effective molecular weight of small PEIs, which are poor vectors compared to the higher molecular weight homologues but less toxic, raises their transfection efficiency due to better DNA binding, we synthesized a library of 144 biodegradable derivatives from two small PEIs and 24 bi- and oligo-acrylate esters. A 423-Da linear PEI and its 1:1 (w/w) mixture with a 1.8-kDa branched PEI were cross-linked with the acrylates at three molar ratios in DMSO. The resulting polymers were screened for their efficiency in delivering a β-galactosidase expressing plasmid to COS-7 monkey kidney cells. Selected most potent polymers from the initial screen were tested for toxicity in A549 human lung cancer cells, and in vivo in a systemic gene delivery model in mice employing a firefly luciferase expressing plasmid.Several polycations that exhibited high potency and low toxicity in vitro were identified from the library. The most potent derivative of the linear 423-Da PEI was that cross-linked with tricycle-[5.2.1.0]-decane-dimethanol diacrylate (diacrylate 14), which exhibited an over 3,600-fold enhancement in efficiency over the parent. The most potent mixed PEI was that cross-linked with ethylene glycol diacrylate (diacrylate 4) which was over 850-fold more efficient than the physically mixed parent PEIs. The relative efficiencies of these polymers were even up to over twice as high as that of the linear 22-kDa PEI, considered the “gold standard” for in vitro and systemic gene delivery. The potent cross-linked polycations identified were also less toxic than the 22-kDa PEI. The optimal vector in vivo was the mixed PEI cross-linked with propylene glycol glycerolate diacrylate (diacrylate 7); it mediated the highest gene expression in the lungs, followed by the spleen, with the expression in the former being 53-fold higher compared to the latter. In contrast, the parent PEIs mediated no gene expression at all under similar conditions, and injection of the polyplexes of the 22-kDa PEI at its optimal N/P of 10 prepared under identical conditions killed half of the mice injected.High-throughput synthesis and transfection assay of a cross-linked library of biodegradable PEIs was proven effective in identifying highly transfecting vectors. The identified vectors exhibited dramatically superior efficiency compared to their parents both in vitro and in an in vivo systemic gene delivery model. The majority of these vectors mediated preferential gene delivery to the lung, and their in vivo toxicity paralleled that in vitro.

A complex formation between hemin and a congruous oligonucleotide not only greatly enhances the former’s peroxidative activity but also results in a biocatalyst (DNAzyme) with a novel specificity. Herein substrate, regio-, enantiomeric, and diastereomeric selectivities of heme, the DNAzyme, and the enzyme horseradish peroxidase are comparatively examined.

High-molecular-mass polyethylenimines (PEIs) are widely used vectors for nucleic acid delivery. We found that removal of the residual N-acyl moieties from commercial linear 25-kDa PEI enhanced its plasmid DNA delivery efficiency 21 times in vitro, as well as 10,000 times in mice with a concomitant 1,500-fold enhancement in lung specificity. Several additional linear PEIs were synthesized by acid-catalyzed hydrolysis of poly(2-ethyl-2-oxazoline), yielding the pure polycations. PEI87 and PEI217 exhibited the highest efficiency in vitro: 115-fold and 6-fold above those of the commercial and deacylated PEI25s, respectively; moreover, PEI87 delivered DNA to mouse lung as efficiently as the pure PEI25 but at a lower concentration and with a 200-fold lung specificity. These improvements stem from an increase in the number of protonatable nitrogens, which presumably results in a tighter condensation of plasmid DNA and a better endosomal escape of the PEI/DNA complexes. As a validation of the potential of such linear, fully deacylated PEIs in gene therapy for lung diseases, systemic delivery in mice of the complexes of a short interfering RNA (siRNA) against a model gene, firefly luciferase, and PEI25 or PEI87 afforded a 77% and 93% suppression of the gene expression in the lungs, respectively. Furthermore, a polyplex of a siRNA against the influenza viral nucleocapsid protein gene and PEI87 resulted in a 94% drop of virus titers in the lungs of influenza-infected animals.

Use of common proteins with no distinctive redox functionalities as chiral templates for asymmetric oxidations and reductions represents an intriguing, but barely explored, phenomenon in biocatalysis. This review focuses on synthetically important and challenging reactions stereoselectively catalyzed or mediated by ubiquitous nonredox proteins, such as bovine serum albumin and chymotrypsin.Graphic

Branched polyethylenimine (PEI) chains with an average molecular mass of 2 kDa (PEI2) have been covalently attached to gold nanoparticles (GNPs), and the potency of the resulting PEI2–GNPs conjugates as vectors for the delivery of plasmid DNA into monkey kidney (COS-7) cells in the presence of serum in vitro has been systematically investigated. The transfection efficiencies vary as a function of the PEI/gold molar ratio in the conjugates, with the best one (PEI2–GNPII) being 12 times more potent than the unmodified polycation. This potency can be further doubled by adding amphiphilic N-dodecyl–PEI2 during complex formation with DNA. The resulting ternary complexes are at least 1 order of magnitude more efficient than the 25-kDa PEI, one of the premier polycationic gene-delivery vectors. Importantly, although unmodified PEI2 transfects just 4% of the cells, PEI2–GNPII transfects 25%, and the PEI2–GNPII/dodecyl–PEI2 ternary complex transfects 50% of the cells. The intracellular trafficking of the DNA complexes of these vectors, monitored by transmission electron microscopy, has detected the complexes in the nucleus <1 h after transfection.

The effect of various chemical modifications of nitrogen atoms on the efficiency of polyethylenimines (PEIs) as synthetic vectors for the delivery of plasmid DNA into monkey kidney cells in vitro has been systematically investigated. The resultant structure–activity relationship has both provided mechanistic insights and led to PEI derivatives with markedly enhanced performance. For example, N-acylation of PEI with the molecular mass of 25 kDa (PEI25, one of the most potent polycationic gene delivery vectors) with alanine nearly doubles its transfection efficiency in the presence of serum and also lowers its toxicity. Furthermore, dodecylation of primary amino groups of 2-kDa PEI yields a nontoxic polycation whose transfection efficiency in the presence of serum is 400 times higher than the parent's and which exceeds 5-fold even that of PEI25.

Poly(4-vinyl-N-alkylpyridinium bromide) was covalently attached to glass slides to create a surface that kills airborne bacteria on contact. The antibacterial properties were assessed by spraying aqueous suspensions of bacterial cells on the surface, followed by air drying and counting the number of cells remaining viable (i.e., capable of growing colonies). Amino glass slides were acylated with acryloyl chloride, copolymerized with 4-vinylpyridine, and N-alkylated with different alkyl bromides (from propyl to hexadecyl). The resultant surfaces, depending on the alkyl group, were able to kill up to 94 ± 4% of Staphylococcus aureus cells sprayed on them. A surface alternatively created by attaching poly(4-vinylpyridine) to a glass slide and alkylating it with hexyl bromide killed 94 ± 3% of the deposited S. aureus cells. On surfaces modified with N-hexylated poly(4-vinylpyridine), the numbers of viable cells of another Gram-positive bacterium, Staphylococcus epidermidis, as well as of the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli, dropped more than 100-fold compared with the original amino glass. In contrast, the number of viable bacterial cells did not decline significantly after spraying on such common materials as ceramics, plastics, metals, and wood.

To circumvent inherent problems associated with pulmonary administration of aqueous-solution and dry-powder protein drugs, inhalation delivery of proteins from their suspensions in absolute ethanol was explored both in vitro and in vivo. Protein suspensions in ethanol of up to 9% (wt/vol) were readily aerosolized with a commercial compressor nebulizer. Experiments with enzymic proteins revealed that nebulization caused no detectable loss of catalytic activity; furthermore, enzyme suspensions in anhydrous ethanol retained their full catalytic activity for at least 3 weeks at room temperature. With the use of Zn2+-insulin, conditions were elaborated that produced submicron protein particles in ethanol suspensions. The latter (insulin/EtOH) afforded respirable-size aerosol particles after nebulization. A 40-min exposure of laboratory rats to 10 mg/ml insulin/EtOH aerosols resulted in a 2-fold drop in the blood glucose level and a marked rise in the serum insulin level. The bioavailability based on estimated deposited lung dose of insulin delivered by inhalation of ethanol suspension aerosols was 33% (relative to an equivalent s.c. injection), i.e., comparable to those observed in rats after inhalation administration of dry powder and aqueous solutions of insulin. Inhalation of ethanol in a relevant amount/time frame resulted in no detectable acute toxic effects on rat lungs or airways, as reflected by the absence of statistically significant inflammatory or allergic responses, damage to the alveolar/capillary barrier, and lysed and/or damaged cells.

Using the plaque reduction assay, relatively simple bicyclic quinone molecules, as well as multiple copies thereof covalently attached to a long polyglutamate‐based polymeric chain, were examined as new inhibitors of various naturally occurring strains of influenza A virus. The polymer‐conjugated inhibitors were found to have a far greater potency (for some as high as two orders of magnitude when a long spacer arm was employed) than their corresponding parent molecules against the human Wuhan influenza strain. However, such polymeric inhibitors failed to exhibit higher potency compared with their small molecule predecessors against the human Puerto Rico and avian turkey influenza strains. These observations, further explored by means of molecular modeling, reveal the previously unrecognized unpredictability of the benefits of multivalency, possibly because of poor accessibility of the viral targets to polymeric agents. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:3896–3905, 2012

By attaching multiple copies of the influenza M2 ion channel inhibitors amantadine (1) and rimantadine (2) to polymeric chains, we endeavored to recover their potency in inhibiting drug-resistant influenza viruses. Depending on loading densities, as well as the nature of the drug, the polymer, and the spacer arm, polymer-conjugated drugs were up to 30-fold more potent inhibitors of drug-resistant strains than their monomeric parents. In particular, a 20% loading density and a short linker group on the negatively charged poly-L-glutamate resulted in one of the most potent inhibitors for 2’s conjugates against drug-resistant influenza strains. Although full recovery of the inhibitory action against drug-resistant strains was not achieved, this study may be a step toward salvaging anti-influenza drugs that are no longer effective. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2450-2459, 2013