General properties of silated hydroxyethylcellulose for potential biomedical applications.

The general properties of hydroxyethylcellulose (HEC) grafted with 3-glycidoxypropyltrimethoxysilane (GPTMS) or 3-glycidoxypropylmethyldiethoxysilane (GPDMS) were studied for potential biomedical applications. The graft involved a Williamson reaction between the free hydroxyl function of HEC and the epoxy function of the two silanes. As the grafted silanes are in ionic form (sodium silanolate), this product remains in gel form at basic pH (>12.3) in aqueous solution. When pH decreases, sodium silanolate is transformed into silanol (2 or 3 silanol functions are carried by silicon, depending on the silane grafted). The silanols interreact, and the gel is transformed into a cross-linking form at room or body temperature. Studies were conducted to optimise this product for specific uses. Steam sterilization was used to compare self-hardening as a function of the silane grafted. Our previous work indicated that HEC grafted with GPTMS has good reactivity, but requires high pH for dissolution, whereas dissolution occurs at lower pH with GPMDS. The rate of silanol condensation for silated HEC was then determined as a function of pH, temperature, type of silane, and the percentage grafted. Condensation rates were ascertained by the viscosity method, and gels were neutralized by different solutions to obtain buffered forms at various pH. The time required to obtain 10(5) mPa x s, with an initial state of 2500 mPa x s, was then calculated. Condensation was catalysed in acid or basic medium at a lower rate at pH 5.5-6.5, and a temperature rise increased the condensation rate, regardless of the pH or silane studied. Silanetriol was more reactive than silanediol. However, as HEC lost considerable viscosity after sterilization, further studies will be conducted to develop new polysaccharides grafted with silane.     

A facile synthesis of azido-terminated heterobifunctional poly(ethylene glycol)s for "click" conjugation

New azido-terminated heterobifunctional poly(ethylene glycol) (PEG) derivatives having primary amine and carboxyl end groups, (Azide-PEG-NH 2 and Azide-PEG-COOH, respectively) were synthesized with high efficiency. An alpha-allyl-omega-hydroxyl PEG was prepared as the first step to Azide-PEG-X (X = NH 2 and COOH) through the ring-opening polymerization of ethylene oxide (EO) with allyl alcohol as an initiator, followed by two-step modification of the hydroxyl end to an azido group. To introduce primary amino or carboxyl functional groups, amination and carboxylation reactions of the allyl terminal ends was then conducted by a radical addition of thiol compounds. Molecular functionalities of both ends of the PEG derivatives thus prepared were characterized by (1)H, (13)C NMR, and MALDI-TOF MS spectra, validating that the reaction proceeded quantitatively. The terminal azido functionality is available to conjugate various ligands with an alkyne group through the 1,3-dipolar cycloaddition reaction condition ("click chemistry").     

Chemoenzymatic construction of a four-component Ugi combinatorial library

The chemoenzymatic preparation of a nine-member Ugi condensation library is described. The carboxylic acid and amine precursors are based on 3-hydroxybutyrate and 4-amino-1-butanol, respectively, and have been acylated selectively using a variety of acyl donors catalyzed by porcine pancreatic lipase. The enzyme is selective for the hydroxyl functionalities on both precursors, thereby yielding 3-acyl-butyric acid and 4-amino-1-acyl compounds. These enzymatically generated derivatives were then subjected to a four-component Ugi condensation reaction in the presence of acetaldehyde and methyl isocyanoacetate. Isolated yields of the alpha-(acylamino)amide Ugi products ranged from 72-95%. The inherent chemoselectivity of enzymatic catalysis may play an increasingly important role in expanding the structural diversity that can be achieved by chemical multicomponent condensation reactions.     

Synthesis and solution behavior of poly(ε-caprolactone) grafted hydroxyethyl cellulose copolymers

Trimethylsilylated hydroxyethyl cellulose (TMSHEC) was synthesized by using hexamethyldisilazane (HMDS) as silylated agent. With the partial protection of hydroxyl groups of HEC by silylation, the novel poly(?-polycaprolactone) (PCL) grafted HEC (HEC-g-PCL) copolymers were successfully prepared by homogenous ring-opening graft polymerization and deprotection procedure. The structure of HEC-g-PCL copolymers was characterized by FTIR and 1H NMR. Fluorescence spectrum of HEC-g-PCL copolymer dilute solution indicated that copolymers could associate and form hydrophobic microdomains in aqueous solution. With the increasing of grafted PCL content, the critical association concentration (cac) of HEC-g-PCL copolymers decreased. The surface tension of HEC-g-PCL copolymers decreased dramatically with the increasing of the concentration and then approached to a plateau value when concentration was above the cac of HEC-g-PCL copolymers. The hydrodynamic radius of the aggregate of copolymer in dilute solution was found to increase with the increasing of the grafted PCL content. When the concentration of copolymer was above the cac, the zero-shear viscosity of the copolymer increased sharply and became much higher than that of HEC at the same concentration.     

Antibacterial surfaces based on polymer brushes: investigation on the influence of brush properties on antimicrobial peptide immobilization and antimicrobial activity

Primary amine containing copolymer, poly(N,N-dimethylacrylamide-co-N-(3-aminopropyl)methacrylamide hydrochloride) (poly(DMA-co-APMA)), brushes were synthesized on Ti surface by surface-initiated atom transfer radical polymerization (SI-ATRP) in aqueous conditions. A series of poly(DMA-co-APMA) copolymer brushes on titanium (Ti) surface with different molecular weights, thicknesses, compositions, and graft densities were synthesized by changing the SI-ATRP reaction conditions. Cysteine-functionalized cationic antimicrobial peptide Tet213 (KRWWKWWRRC) was conjugated to the copolymers brushes using a maleimide-thiol addition reaction after initial modification of the grafted chains using 3-maleimidopropionic acid N-hydroxysuccinimide ester. The modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, atomic force microscopy (AFM), and ellipsometry analysis. The conjugation of the Tet213 onto brushes strongly depended on graft density of the brushes at different copolymer brush compositions. The peptide density (peptides/nm(2)) on the surface varied with the initial composition of the copolymer brushes. Higher graft density of the brushes generated high peptide density (pepetide/nm(2)) and lower number of peptides/polymer chain and vice versa. The peptide density and graft density of the chains on surface greatly influenced the antimicrobial activity of peptide grafted polymer brushes against Pseudomonas aeruginosa.     

Silicon Composite Electrodes with Dynamic Ionic Bonding

Silicon (Si) composite electrodes are developed with increased cycle lifetimes and reliability through dynamic ionic bonding between active Si nanoparticles and a polymer binder. Amine groups are covalently attached to Si nanoparticles via surface functionalization. Si composite electrodes are fabricated by combining the Si nanoparticles with a poly(acrylic acid) (PAA) binder. The formation of ionic bonds between amine groups on Si particles and carboxylic acid groups on the PAA binder is characterized by X‐ray photoelectron spectroscopy and Raman spectroscopy. Si composite anodes with ionic bonding demonstrate long term cycling stability with capacity retention of 80% at 400 cycles at a current density of 2.1 A g^?1 and good rate capability. The dynamic ionic bonds effectively mitigate the deterioration of electrical interfaces in the composite anodes as suggested by stable impedance over 300 cycles.     

Poly(glycidylmethacrylate) brushes generated on poly(VBC) beads by SI-ATRP technique: Hydrazine and amino groups functionalized for invertase adsorption and purification

Crosslinked-poly(vinylbenzylchloride), poly(VBC), beads were prepared by suspension polymerization and poly(glycidylmethacrylate) was grafted by surface-initiated-atom radical polymerization (SI-ATRP) technique. Epoxy groups of the grafted poly(GMA) were reacted with hydrazine and ammonia to create an affinity binding sites. The hydrazine and amine functionalized poly(VBC-g-GMA) beads were used as an affinity support for adsorption of invertase from solution and yeast crude extract. The influence of pH, equilibrium time, ionic strength and initial invertase concentration on the adsorption capacities of both hydrazine and amine functionalized beads has been investigated. Maximum invertase adsorptions onto hydrazine and amine functionalized beads, were 86.7 and 30.4 mg/g at pH 4.0 and 5.5, respectively. The experimental equilibrium data fitted well to the Temkin isotherm model. Finally, the hydrazine functionalized poly(VBC-g-GMA) beads were used for the purification of invertase from crude yeast extract in a batch system and the purity of the eluted invertase from the hydrazine functionalized beads was determined as 92% by HPLC from single step purification protocol.     

Facile synthetic procedure for omega, primary amine functionalization directly in water for subsequent fluorescent labeling and potential bioconjugation of RAFT-synthesized (co)polymers

We describe a facile method to amine functionalize and subsequently fluorescently label polymethacrylamides synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. RAFT-generated poly(N-(2-hydroxypropyl) methacrylamide-b-N-[3-(dimethylamino)propyl] methacrylamide) (poly(HPMA-b-DMAPMA)), a water soluble biocompatible polymer, is first converted to a polymeric thiol and functionalized with a primary amine through a disulfide exchange reaction with cystamine and subsequently reacted with the amine-functionalized fluorescent dye, 6-(fluorescein-5-carboxamido)hexanoic acid, succinimidyl ester (5-SFX). Poly(HPMA258-b-DMAPMA13) (Mn = 39 700 g/mol, Mw/Mn = 1.06), previously synthesized by RAFT polymerization, was used to demonstrate this facile labeling method. The problem with labeling the omega-terminal chain end of a RAFT-synthesized polymethacrylamide is that the reduced end yields a tertiary thiol with low reactivity. The key to labeling poly(HPMA-b-DMAPMA) is to first reduce the dithioester chain end with a strong reducing agent such as NaBH4, and then functionalize the tertiary polymeric thiol with a primary amine through a disulfide exchange reaction with dihydrochloride cystamine. We show that the disulfide exchange reaction is efficient and that the amine-functionalized poly(HPMA-b-DMAPMA) can be easily labeled with the fluorescent dye, 5-SFX. This concept is proven by using a ninhydrin assay to detect primary amines and UV-vis spectroscopy to measure the degree of conjugation.     

Degradable-brushed pHEMA-pDMAEMA synthesized via ATRP and click chemistry for gene delivery

Brushed polymers composed of a backbone of poly(hydroxyethyl methacrylate) (pHEMA) onto which poly(2-(dimethylamino)ethyl methacrylate)s (pDMAEMAs) was grafted via a hydrolyzable linker were synthesized and evaluated as nonviral gene delivery vectors. Both pDMAEMA and pHEMA polymers with controlled molecular weights and narrow distributions were synthesized by controlled atom transfer radical polymerization (ATRP). The azide initiator was used to ensure complete and monoazide functionalization of the pDMAEMA polymer chains. Click reaction between pHEMA with alkyne side groups and the azide end group in the pDMAEMA resulted in a high-molecular-weight polymer composed of low-molecular-weight constituents via an easily degradable carbonate ester linker. The length of the pDMAEMA grafts as well as the number of grafts of the brushed pHEMA-pDMAEMA can be easily varied. At physiological conditions (pH 7.4 and 37 degrees C), the brushed polymer degraded by hydrolysis of the carbonate ester with a half-life of 96 h. The molecular weights of the formed degradation products was very close to that of the starting pDMAEMA, which is likely below the renal excretion limit (<30 kDa). It was shown that the degradable brushed pHEMA-pDMAEMAs were able to condense plasmid DNA into positively charged nanosized particles. The resulting polyplexes were able to transfect cells efficiently in the presence of the endosomal membrane disrupting INF-7 peptide, and all these degradable polymers showed lower cellular toxicity compared to a high-molecular-weight pDMAEMA reference. On the other hand, the low-molecular-weight pDMAEMA used for the grafting to pHEMA was neither able to condense the structure of DNA nor able to transfect cells. This study demonstrates that grafting a low-molecular-weight cationic polymer via a hydrolyzable linker to a neutral hydrophilic polymer is an effective approach to modulate the transfection activity and toxicity profile of gene delivery polymers.     

Enzymatic grafting of a natural product onto chitosan to confer water solubility under basic conditions

Chitosan is a natural biopolymer whose rich amine functionality confers water solubility at low pH. At higher pH's (greater than 6. 5), the amines are deprotonated and chitosan is insoluble. To attain water solubility under basic conditions we enzymatically grafted the hydrophilic compound chlorogenic acid onto chitosan. Despite its name, chlorogenic acid is a nonchlorinated phenolic natural product that has carboxylic acid and hydroxyl functionality. The enzyme in this study was tyrosinase, which converts a wide range of phenolic substrates into electrophilic o-quinones. The o-quinones are freely diffusible and can undergo reaction with the nucleophilic amino groups of chitosan. Using slightly acidic conditions (pH = 6), it was possible to modify chitosan under homogeneous conditions. When the amount of chlorogenic acid used in the modification reaction exceeded 30% relative to chitosan's amino groups, the modified chitosan was observed to be soluble under both acidic and basic conditions, and to have a pH window of insolubility at near neutral pH. 1H NMR spectra confirmed that chitosan was chemically modified, although the degree of modification was low. Copyright 1999 John Wiley & Sons, Inc.     

Preparation and characterization of poly(acrylic acid)-hydroxyethyl cellulose graft copolymer

Poly(acrylic acid) hydroxyethyl cellulose [poly(AA)-HEC] graft copolymer was prepared by polymerizing acrylic acid (AA) with hydroxyethyl cellulose (HEC) using potassium bromate/thiourea dioxide (KBrO(3)/TUD) as redox initiation system. The polymerization reaction was carried out under a variety of conditions including concentrations of AA, KBrO(3) and TUD, material to liquor ratio and polymerization temperature. The polymerization reaction was monitored by withdrawing samples from the reaction medium and measuring the total conversion. The rheological properties of the poly(AA)-HEC graft copolymer were investigated. The total conversion and rheological properties of the graft copolymer depended on the ratio of KBrO(3) to TUD and on acrylic acid concentration as well as temperature and material to liquor ratio. Optimum conditions of the graft copolymer preparation were 30mmol KBrO(3) and 30mmol TUD/100g HEC, 100% AA (based on weight of HEC), duration 2h at temperature 50°C using a material to liquor ratio of 1:10.     

Oligonucleotides containing 7-vinyl-7-deazaguanine as a facile strategy for expanding the functional diversity of DNA

A modified nucleobase 7-vinyl-7-deazaguanine ((V)G) produced adducts with maleimides through Diels-Alder cycloaddition under very mild conditions. By this method, post-synthetic modification to oligonucleotides with diverse functionality (carboxylic acid, pyrene, benzophenone, succinimidyl ester, nitroxide and biotin) was accomplished.     

Bacillus cereus phospholipase C: carboxylic acid ester specificity and stereoselectivity

Thiophosphate analogs of phosphatidylcholine have been synthesized with varying structural complexity. These analogs have been used in a continuous spectrophotometric assay for phospholipase C (Bacillus cereus) to estimate the minimal structural requirements associated with the non-polar portion of the substrate phospholipid. The analogs were of three types containing zero, one or two carboxylic acid ester functionalities. The analogs with one or two ester groups acted as substrates for phospholipase C, while those without an ester functionality were not hydrolyzed. The rac-phosphatidylcholine analog with two ester functionalities gave biphasic time-course results, and was subsequently resolved into enantiomers by selective hydrolysis with a sterospecific phospholipase A2 (Crotalus atrox). The enantiomer with R absolute configuration was rapidly hydrolyzed by the phospholipase C while the enantiomer with the S configuration was slowly hydrolyzed after a long induction period. The results suggest that the B. cereus phospholipase C is specific for an ester functionality and is stereoselective for the R absolute configuration at glycerol C-2.     

Cell attachment and long-term growth on derivatizable polyacrylamide surfaces

Important cellular characteristics, including selective adhesion, growth rate, motility, and differentiation, are controlled, in part, by signals received at the cell surface. The molecular mechanisms for the cell surface control of these cell behaviors are largely unknown. In order to probe the role of specific extracellular molecules in controlling cell function, we report the development of synthetic surfaces which generally support the long-term growth of cells yet can be readily derivatized with a wide variety of molecules of biological interest. Polyacrylamide gels containing a gradient of active ester groups were prepared and then the esters were displaced with ligands to generate a gradient of carboxylic acid, tertiary amine, or hydroxyl groups. When untransformed mouse fibroblasts (BALB/3T3) were seeded on the various surfaces, they attached and grew only on those derivatized with carboxylic acids or hydroxyl groups within narrow concentration ranges. Cell growth rate, density, and morphology on polyacrylamide gels containing the optimal concentration of carboxylic acid groups (approximately 30 mumol/ml) were comparable to those on tissue culture plastic, whereas growth on hydroxyl group-derivatized gels was less extensive. In contrast, short-term (90-min) adhesion to hydroxyl group-derivatized gels was greater than that to carboxylic acid-derivatized gels. Both short-term adhesion and long-term growth required serum. Growth-supportive polyacrylamide gels were readily derivatized with molecules of biological interest. The techniques reported here are applicable to other types of cell in culture since the nature and concentration of substratum functional groups can be easily varied and tested for support of long-term cell growth.     

Solid phase and solution synthesis of NvocLys(CO(CH<Subscript>2</Subscript>)<Subscript>5</Subscript>NH–NBD)OCH<Subscript>2</Subscript>CN,a trifunctional fluorescent lysine derivative

Herein, we describe a general strategy for the facile synthesis of a multifunctional amino acid derivative bearing both fluorescent and photolabile groups such as the lysine derivative NvocLys(CO(CH₂)₅NH–NBD)OCH₂CN (1) that can be used as a biophysical tool for studying protein structure. The synthetic strategy involves functionalization of the amine groups while the amino acid is attached to a solid support, followed by esterification of the carboxylic acid in solution. The solid support protects the caboxylic acid, preventing a side reaction associated with the synthesis in solution and obviating the need for chromatographic purification of several intermediates. This synthetic strategy can be used for the preparation of a variety of amino acid derivatives with unusual α-amine and side chain functionalities.     

Hyperbranched poly(amino ester)s with different terminal amine groups for DNA delivery

Hyperbranched poly(amino ester)s containing tertiary amines in the core and primary, secondary, and tertiary amines in the periphery, respectively, were evaluated for DNA delivery in vitro. The same core structure facilitated the investigation on the effects of the terminal amine type on the properties of hyperbranched poly(amino ester)s for DNA delivery. The hydrolysis of the poly(amino ester)s was monitored using (1)H NMR. The results reflected that the terminal amine type had negligible effects on the hydrolysis rate but was much slower than that of linear poly(amino ester)s, probably due to the compact hyperbranched spatial structure preventing the accessibility of water. In comparison with PEI 25 K, the hyperbranched poly(amino ester)s showed much lower cytotoxicity in Cos7, HEK293, and HepG2 cells. Gel electrophoresis indicated that poly(amino ester)s could condense DNA efficiently, and the zeta potentials and sizes of the complexes formed with different weight ratios of hyperbranched poly(amino ester)s and DNA were measured. Remarkably, all the hyperbranched poly(amino ester)s showed DNA transfection efficiency comparable to PEI 25 K in Cos7, HEK293, and HepG2 cells regardless of the terminal amine type. Therefore, the terminal amine type had insignificant effects on the hydrolysis rate, cytotoxicity, DNA condensation capability, and in vitro DNA transfection efficiency of the hyperbranched poly(amino ester)s.     

Protonated polynucleotide structures. IX. Disproportionation of poly (G)-poly (C) in acid medium

The interaction between poly (G) and poly (C) was investigated in neutral and acid medium by optical methods. Three main points arise from this investigation. (1) The formation of poly (G)·poly (C) was complete only above an ionic strength of about 0.6M [Na⁺]. Lowering the ionic strength increased the amounts of free poly (G) and free poly (C) that could be detected. (2) When titrating towards acid pH values a transition took place which was characterized by potentiometry, mixing curves, and circular dichroism: a three-stranded poly (G)·poly (C)·poly (C⁺) complex was formed analogous to the transition observed for the acid titration of poly (I)·poly (C). (3) Even when the poly (G)·poly (C) complex was incompletely formed (at low ionic strength) in neutral medium all poly (C) entered the triple-stranded complex.     

Synthesis and evaluation of triazole linked glycosylated 18β-glycyrrhetinic acid derivatives as anticancer agents

A series of glycosyl triazol linked 18β-glycyrrhetinic acid (GA) derivatives have been synthesized using 1,3-dipolar cycloaddition reaction of per-O-acetylated glycosyl azide derivatives (4a–h) with propargyl ester of 18β-glycyrrhetinic acid (GA) (2 and 3) following the concept of ‘Click chemistry’. The synthesized triazole derivatives were de-O-acetylated to furnish compounds (7a–h and 8a–c) with free hydroxyl groups in the carbohydrate moieties, which were evaluated for their anticancer potential against human cervical cancer cells (HeLa) and normal kidney epithelial (NKE) cells. GA (1), compound 7d, compound 7g and compound 8c showed promising anticancer activities.     

Grafting of poly[2-(tert-butylamino)ethyl methacrylate] onto polypropylene by reactive blending and antibacterial activity of the copolymer

To combine low cost, good mechanical properties, and antibacterial activity in one material, a nonquaternized polymeric biocide, i.e., poly[2-(tert-butylamino)ethyl methacrylate] (PTBAEMA), was dispersed within a commodity plastic, i.e., polypropylene (PP). The high immiscibility of the two polymers was tackled by reactive compatibilization and thus by reaction of commercially available maleic anhydride grafted polypropylene with primary amine-end-capped PTBAEMA. This reactive polymethacrylate was synthesized by atom-transfer radical polymerization with an azide-containing initiator. The azide end group was converted into a primary amine by the Huisgen [3 + 2] cycloaddition of propargylamine. The accordingly formed PP-g-PTBAEMA copolymer was melt dispersed within neat PP and processed as fibers, whose antimicrobial properties were assessed by the viable cell counting method against Escherichia coli. The antibacterial activity was long-lasting as a result of the anchoring of the PTBAEMA chains onto PP, which prevented them from being released from the surface of the fibers.     

Engineered Si(1 0 0) surfaces for the gas-phase anchoring of metal β-diketonate complexes

A synthetic strategy for the covalent anchoring of nickel β-diketonate complexes on Si(1 0 0) has been examined. Engineered Si(1 0 0) surfaces were prepared by the Si-grafting of 10-undecylenic acid methyl ester followed by hydrolysis of the ester to free the carboxylic functions suited for the anchoring of the Ni complex. Bis(pentane-2,4-dionate)Ni(II) was bonded to the functionalized surface from the gas phase by the exchange of the acetylacetonate ligand with the grafted acid. The surface density of the anchored Ni complex was controlled by tuning the surface concentration of carboxylic groups adopting a mixed monolayer of undecylenic acid and 1-decene used as a spectator spacer. The nickel decorated silicon surfaces were characterized by attenuate total reflectance infrared absorption spectroscopy (ATR-IRAS) and angle resolved X-ray photoelectron spectroscopy (AR-XPS).