Quantification of the complexation of protein with neutral water borne polymer by fluorescence spectroscopy

The complexation of bovine serum albumin (BSA) with poly(N-isopropylacrylamide) (PNIPAM) in an aqueous system (pH 7) has been investigated by means of fluorescence spectroscopy. Through analyzing the change of fluorescence intensity of protein chromophores caused by complexation, a quantitative mathematical equation has been established and the average number of bound proteins per neutral polymer (n_b) can be calculated accurately without destroying the dynamic equilibrium of aqueous complex system. At the same time, with the help of this equation, the extreme value of n_b can also be calculated when PNIPAM concentration is low enough. Compared with traditional calculation methods, this method has the advantages of rapid detectability, high sensitivity, accuracy and extensive applicability. Thus, it is a better way to study the complexation of protein with polymer.     

Optimization of protein encapsulation in PLGA nanoparticles

Influences of process parameters were investigated on the efficiency of encapsulation of bovine serum albumin (BSA) in poly(dl-lactic-co-glycolic acid) (PLGA) nanoparticles produced by w₁/o/w₂ (water-in-oil-in-water) double emulsion-solvent evaporation method. According to a 5-factorial 3-level Box-Behnken type experimental design aqueous solution of BSA was emulsified in an immiscible organic phase composed of dichloromethane and various quantities of dissolved PLGA to get water-in-oil (w₁/o) emulsion. This latter was then dispersed in a second aqueous phase (w₂) containing poly-vinyl-alcohol (PVA) surfactant as an emulsifier/stabilising agent. PLGA nanoparticles with encapsulated BSA were obtained by evaporating the dichloromethane from the w₁/o droplets. Encapsulation efficiency was determined as the weight ratio of BSA remained in the PLGA nanoparticles relative to the total weight of BSA used in the process. By statistical evaluation of the experimental results an equation was proposed to predict the encapsulation efficiency as a function of five process variables. Two optimization procedures were carried out to increase the efficiency of encapsulation, with and without constraints referring to the required mean particle size. Correlation was found between the latter and the achievable maximal encapsulation efficiency under optimal process conditions.     

Change in the molecular weight distribution of poly(ethylene oxide) caused by the complexation with poly(acrylic acid)

The complexation between poly(ethylene oxide) (PEO) and poly(acrylic acid) (PAA) was made by using double the molar quantity of either polymer component at pH 2 where the resulting complex completely precipitates. After the removal of the precipitate, PEO or PAA remaining in the supernatant was subjected to gel permeation chromatography to investigate the change in the molecular weight distribution (MWD) caused by the complexation. No remarkable difference is observed in the MWD curves for PEO[1] (M_w=1.37 × 10⁴) before and after the complexation with PAA[1] (M_w=1.10 × 10³) and PAA[2] (M_w=4.16 × 10⁵). However, the MWD curves of PEO[2] (M_w=1.26 × 10⁵) and PAA[2] become shortened and shift to the low molecular weight side after the complexation with PAA[1] or [2] and PEO[2], respectively. This tendency is enhanced by increasing the complexation temperature. From these results, it is indicated that the complexation between PEO and PAA deals with an equilibrium reaction, and the equilibrium constant is dependent on the chain length of both polymer components and also on the complexation temperature.     

Macroinorganics: 8. Chelation of copper(II) ion with some new poly(amido-amines)

The effect on Cu(ll) complexing ability of the number (n) of methylenic groups between aminic nitrogens has been studied for two classes of poly (amido-amines). In order to better elucidate the mechanism of complex formation, low molecular weight models of the polymers have also been studied. The complexing abilities of polymers and models are very similar and decrease by increasing n. Indeed compounds with n = 4 fail to form complexes in aqueous solution. Some viscosimetric titrations have been performed in order to ascertain possible conformational transitions upon complexation.     

High performance liquid chromatography study of water‐soluble complexes and covalent conjugates of polyacrylic acid with bovine serum albumin

Polycomplex formation of bovine serum albumin (BSA) and polyacrylic acid (PAA) was studied by pH titration, fluorescence, and HPLC methods in water solutions. It was shown that the complex formation and the solubility (phase state) of the polycomplexes depended on pH of the solutions and ratio of the components. The stability of PAA‐BSA complexes was negligibly weak when pH = 6.0–7.0 [pH > pI (isoelectric pH)]. Stable water‐soluble polycomplexes formed at pH = pI (pH 5.0) in a wide range of n_BSA/n_PAA ratios (0.05–50) and coexisted with free protein molecules at the higher ratios of components. Existence of water‐soluble and insoluble PAA‐BSA complexes has been observed at pH ≤ 4.5. The soluble to insoluble state transition of polycomplexes has been confirmed and binding mode of polyelectrolytes to proteins was assumed dependent on the ratio of components as a result of formation of soluble polycomplexes, complex coacervation, or amorphous precipitates. Soluble complexes were formed as fully homogenized mixtures. Fraction composition of the mixtures and insoluble complexes depended on the protein/polymer ratio and over the critical protein/polymer ratio, the soluble polycomplexes coexisted with free BSA molecules. On the other hand, the comparision of covalent conjugate formation and complexation of PAA with BSA has been revealed. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

Temperature-induced thickening of sodium carboxymethylcellulose and poly(N-isopropylacrylamide) physical blends in aqueous solution

This study describes the synthesis of poly(N-isopropylacrylamide) (PNIPAM) via free radical polymerization, the preparation of physical blends containing sodium carboxymethylcellulose (CMC) and PNIPAM in aqueous solution, at total polymer concentrations of 2 and 6?g/L in different compositions, and applies rheology to investigate interactions between PNIPAM and CMC compared to pure polymers, in aqueous solution. Rheological measurements indicated thermothickening behavior for the 50?% PNIPAM–50?% CMC physical blend in aqueous solution, at 6?g/L, as viscosity rose when temperature was increased to a range of 25–40?°C. Similar thermothickening behavior was observed for the 25?% CMC–75?% PNIPAM physical blend in solution, at a total polymer concentration of 2?g/L. These results provide new information for preparing physical blends in aqueous solutions exhibiting thermothickening behavior, indicating that this behavior depends on total polymer concentration and composition of the mixture.     

Complexation Equilibrium Constants of Poly(vinyl alcohol)-Borax Dilute Aqueous Solutions – Consideration of Electrostatic Charge Repulsion and Free Ions Charge Shielding Effect

Equilibrium dialysis experiments of dilute poly(vinyl alcohol) (PVA) in borax aqueous solution (PVA concentration was 2 g/L and borax concentration ranged from 0.0 M to 0.05 M) were carried out to determine the concentration of free borate ions in equilibrium with the PVA-borate complex at 25°C, and hence the amount of borate bound to PVA. The binding isotherm of borate ion on PVA molecules was investigated. The concentration of borate ion bound on PVA was found to increase with increasing borax concentration and reached an asymptote at high borax concentrations. The complexation equilibrium constant between borate and PVA diol was found to decrease initially then increases and finally decreases with increasing borax concentration while PVA concentration was fixed in a constant value. The equlibrium constant of free borate ion with borate-PVA diol complex was a balance result of excluded volume of PVA molecular chains, electrostatic charge repulsion between free borate ions and borate-PVA diol complexes, and electro-shielding of free Na⁺ ions on negatively charged PVA-borate complexes. In this study, the electrostatic charge repulsion between free borate ions and borate-PVA diol complex ions and free Na⁺ ions shielding effect are considered in the estimation of equilibrium constants of PVA diol-borate complexation. A fixed value of equilibrium constant is obtained with increasing borax concentration while borax concentraion is below a critical concentration [borax]_crt. As borax cocentration is higher than [borax]_crt, a lots of PVA diols form complexes with borate ions and a high shielding of free Na⁺ ions on negatively charged borate-PVA diol complexes causes the PVA-borate complexes to behave like neutral polymers. Few free PVA diol binding sites are available for reaction with free borate ions while borax concentration is higher than [borax]_crt resulting in a decrease in equilibrium constant with increasing borax concentration.     

Tailorable polyelectrolyte protein complex based on poly(aspartic acid) and bovine serum albumin

Protein–polyelectrolyte complexes (PPC) are playing an important role in a variety of chemical and biological processes, such as protein separation, enzyme stabilization, and polymer drug delivery. The present investigation is focused on evaluation of the PPC formation between a synthetic polypeptide (poly(aspartic acid) – PAS) and a natural protein (bovine serum albumin – BSA). The PPC obtained from PAS and BSA in different ratio was investigated by corroboration of various techniques of characterization as: spectroscopy, microscopy, thermogravimetric analysis, dynamic light scattering (DLS), and zeta potential determination, measurements which were performed in static and/or dynamic conditions. The static contact angle of the sample films was also determined in order to evaluate the changes brought upon surface free energy of the prepared PPCs in interdependence with the complexes composition. The critical conditions for complex formation in BSA–polyelectrolyte systems were underlined. The phenomenon was better evidenced in case of a ratio of 1/1 wt between the two polymers, both in static and/or dynamic conditions.     

Coating of gold nanoparticles by thermosensitive poly(N-isopropylacrylamide) end-capped by biotin

Gold nanoparticles (NPs) were prepared by reduction of HAuCl₄ in aqueous solution and stabilized by poly(N-isopropylacrylamide) (PNIPAM). PNIPAM was prepared by two distinct routes: (i) conventional free-radical polymerization leading to polymer without any reactive end-group, and (ii) Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization with 2-dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionic acid (DMP) as a RAFT agent. PNIPAM with low polydispersity was then end-capped by an α-carboxylic acid and an ω-trithiocarbonate that was converted into an ω-thiol upon hydrolysis. This hetero-telechelic polymer was analyzed by mass spectroscopy, size exclusion chromatography (SEC) and ¹H NMR. Even without thiol end-group, known for chemisorption onto gold, PNIPAM was effective in stabilizing gold NPs (∼1-5 nm). The thermosensitivity of PNIPAM at the surface of gold NPs was, however, dependent on the molecular weight of the chains. Finally, the α-carboxyl end-group of PNIPAM was used to anchor biotin, which is indeed known for complexation with avidin, which is a possible strategy for the coated gold NPs to be involved as building blocks in supramolecular assemblies. TEM and UV-vis spectroscopy were used to characterize the gold nanoparticles.     

Interruption of hydration state of thermoresponsive polymer,poly(N-isopropylacrylamide) in guanidinium hydrochloride

In this article, we investigate the effect of guanidinium hydrochloride (GdnHCl) on the phase transition temperature of poly (N-isopropylacrylamide) (PNIPAM) in aqueous solution with the aid of fluorescence spectroscopy, viscosity (η), and dynamic light scattering (DLS) measurements. Temperature dependent fluorescence intensity measurements have been employed successfully to determine the conformational change of PNIPAM through evaluating the Gibbs free energy changes. Additionally, direct hydrogen bonding formation between GdnHCl and PNIPAM has been confirmed by Fourier transform infrared (FTIR) spectroscopy measurements at various concentrations of GdnHCl. The intermolecular interactions were also studied in terms of amide I band analysis, which reveals the interruption of hydrated coil conformation of PNIPAM. The degree of destabilization of PNIPAM progressively increases with increasing concentration of the denaturant.     

Chronopotentiometric potential-time curves of electron transfer across the interface between two immiscible electrolyte solutions

Theoretical potential-time curves were derived that are mathematical expressions of the electron transfer reaction O1 (w) + R2(non) ? R1(w) + O2(non) proceeding at the interface of two immiscible phases between the redox couple O1/R1 in the aqueous phase (w) and the redox couple O2/R2 in the non-aqueous phase (non) under galvanostatic conditions. Procedures for the determination of the diffusion coefficients of the reactants in the corresponding phase and for the evaluation of thermodynamic and kinetic parameters of the electron transfer reaction from the potential-time curves were suggested.     

Molecular characterization of α,β-poly(N-2-hydroxyethyl)-dl-aspartamide derivatives as potential self-assembling copolymers forming polymeric micelles

A family of graft copolymers derivatives obtained from α,β-poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA) have been studied as potential self-assembling macromolecules forming stable polymeric micelles at low critical micellar concentration. These polymers are obtained grafting on PHEA poly(ethylene glycol) (PEG) (M_w 5000 g/mol) (PHEA-PEG), hexadecylamine (PHEA-C₁₆) or both moieties (PHEA-PEG-C₁₆). The PHEA derivatives were characterised by a multi-angle light scattering (MALS) photometer on line to a size exclusion chromatography system in obtaining the molar mass distribution of the polymers. In addition, to investigate the capacity to form micellar aggregates in aqueous medium the MALS photometer was used in off-line batch mode in obtaining molar mass and dimension of the polymeric aggregates.     

Dye-Affinity Partitioning of Acidic,Basic, and Neutral Proteins in Ionic Liquid-Based Aqueous Biphasic Systems

The partitioning behavior of model acidic (bovine serum albumin, BSA), basic (hen egg white lysozyme, LYS), and neutral (horse heart myoglobin, MYO) proteins in ionic liquid-based aqueous biphasic systems containing Reactive Red-120 dye has been reported. This study found that partition coefficients of the proteins depended on their chemical structures, pH of the aqueous phase, temperature, and composition of the aqueous biphasic system. It was found that partition coefficients of proteins in aqueous biphasic system containing RR-120 were in the order of LYS > MYO > BSA. Recovery of the hydrophilic ionic liquid, [C₄mim]Br, was achieved by using hydrophobic ionic liquid, [C₄mim]PF_6.     

Smart organic-inorganic nanohybrid stars based on star-shaped poly(acrylic acid) and functional silsesquioxane nanoparticles

pH- and salinity-responsive organic-inorganic nanohybrid stars based on poly(acrylic acid) (PAA) stars and N,N-di(2,3-dihydroxypropyl)3-aminopropylfunctional silsesquioxane nanoparticles are readily formed by mixing of aqueous solutions of the components. The interaction between stars of two different arm lengths, (PAA₁₀₀)₂₁, (PAA₂₀₀)₂₄, with water-soluble silsesquioxane nanoparticles is studied according to changes in pH and salt concentration. The original size of the stars is conserved during complexation according to dynamic light scattering (DLS) measurements and light scattering (LS) titration experiments, which exclude star-star aggregation or crosslinking during the interaction. The proposed interaction mechanism is based on hydrogen-bonding and Coulomb interactions. Cryogenic transmission electron microscopy measurements demonstrate the formation of nanohybrid stars. Small-angle neutron scattering experiments enable a quantitative determination of the fraction of bound nanoparticles and indicate an equilibrium between free and bound nanoparticles.     

Study of specific interactions in inclusion complexes of amine-terminated PAMAM dendrimer/flavonoids by experimental and computational methods

Polyamidoamine (PAMAM) dendrimers have a multifunctional structure, able to encapsulate molecules for pharmacological applications. We evaluated the specific interaction that govern the encapsulating and affinity of one group of natural and synthetic flavonoids into the G₅-PAMAM dendrimers. The complexation and capture percent of one flavonoid series into G₅-PAMAM dendrimers, under neutral and acid pH conditions, were studied through UV–Vis spectroscopy. Additionally, only three of the flavonoids (two synthetic and one natural) were studied by high-performance liquid chromatography (HPLC) and molecular dynamic (MD) simulation, at neutral pH to calculate the affinity constants (K_d) and binding free energies (ΔG_b). From spectroscopic results, we observed that the encapsulation was much more rapid at low pH than at neutral pH, which was attributed to a greater number of cavities inside the dendrimer. The MD simulations suggested that the more compact molecular structure at neutral pH reduces the capture kinetics. Finally, the relative binding free energies calculated using MD simulations showed the same tendency as the experimental data for the three complexes. These affinities appear to be due to a complex balance of different contributions, which cannot be attributed to hydrogen bonds or charge–charge interactions alone. Nevertheless, we suggest that a protocol including UV–Vis, HPLC, and MD simulation can be a powerful predictive tool to determine the affinity of drug binding to nanocarriers.     

Synthesis, reactivity, and pH-responsive assembly of new double hydrophilic block copolymers of carboxymethyldextran and poly(ethylene glycol)

Double hydrophilic block copolymers (DHBC) were prepared by end-to-end coupling of two biocompatible water-soluble homopolymers: the polysaccharide dextran (M_w 8300 or 14,700 g mol⁻¹) and ω-amino poly(ethylene glycol) (PEG-NH₂, M_w 3000 or 7000 g mol⁻¹). The synthesis involved, first, specific oxidation of the dextran terminal aldehyde group and, second, covalent linkage of PEG-NH₂ via a lactone aminolysis reaction. The diblock copolymers dextran-PEG (DEX-PEG) were converted in high yield into the corresponding carboxymethyldextran-PEG (CMD-PEG) derivatives with control over the degree of substitution, from 30 to 85 mol% CH₂COOH groups per glucopyranosyl unit. Further modifications of a CMD-PEG block copolymer led to N-(2-aminoethyl)carbamidomethyldextran-PEG yielding a pair of oppositely-charged DHBC of identical charge density, chain length, and neutral block/charged block content. The properties of CMD-PEG in aqueous solutions were studied by static and dynamic light scattering as a function of solution pH, providing evidence of the pH-sensitive assembly of the copolymers driven by inter- and intra-chain hydrogen-bond formation.     

NMR paramagnetic probing of polymer solutions using manganese(II) ions

Nuclear magnetic relaxation studies show that manganese(II) ions can serve as paramagnetic probes for investigation of aqueous solutions of polyelectrolytes (polystyrene sulfonate and polyethylenimine) and their mixtures. The analysis of the measured rates of spin–lattice (R ₁) and spin–spin (R ₂) relaxations reveals the differences in the binding character of manganese(II) ions with polystyrene sulfonate and polyethylenimine. In a mixture of the polymers in acidic and neutral media, manganese(II) forms two types of ternary complexes. Using the suggested method for definition of the hydration degree of a coordination sphere of the bound probe ions, the number of water molecules in the first sphere of the polymer-bound manganese(II) ions is found on the basis of the value of the R ₂/R ₁ ratio.     

Influence of different treatments on characteristics of nanooxide powders alone or with adsorbed polar polymers or proteins

Morphological and structural characteristics of a variety of powder nanocomposites with fumed oxides (silica, silica/alumina, silica/titania) and different linear polar polymers (poly(ethylene oxide), poly(ethylene glycol), poly(vinyl alcohol), poly(vinyl pyrrolidone), polydimethylsiloxane and proteins (ossein, gelatine, BSA)), were analyzed using nitrogen adsorption, infrared (IR) spectroscopy, AFM, and TPD MS methods. A monolayer or lower coverage of oxide nanoparticles by linear polar polymers results in relatively small changes in the specific surface area and adsorption capacity compared with similarly treated fumed oxides alone. The pore size distributions of dried solid residual oxide/polymer samples demonstrate a more ordered pore structure than the initial powders. This structure, as well as the morphology of secondary particles, depends slightly on the content of polymers (C_pol) at coverage less than a monolayer. If polymer-polymer interactions are weaker than polymer-oxide interactions, the perturbation degree Ф (normalized to molecular weight of a polymer segment m_seg) of surface silanols at fumed silica A-300 depends very weakly on the type of adsorbed polymers. Among the studied polymers only PVA demonstrates Ф(C_pol/m_seg) values smaller than the other systems because of the formation of strong hydrogen bonds between PVA molecules.     

Polymer-Decorated Cellulose Nanocrystals as Environmentally Friendly Additives for Olefin-Based Drilling Fluids

In this study, we intended to evaluate the performance of olefin-based drilling fluids after addition of cellulose nanocrystal (CNC) derivatives. For this purpose, firstly, cellulose nanocrystals, produced from sulfuric acid hydrolysis of cotton fibers, were functionalized with poly(N-isopropylacrylamide) (PNIPAM) chains via free radicals. The samples were then characterized via Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), confocal microscopy, dynamic light scattering (DLS), and zeta potential measurements in water. The FTIR and NMR spectra exhibited the characteristic signals of CNC and PNIPAM groups, indicating successful grafting. As expected, X-ray diffractograms showed that the crystallinity of CNCs reduces after chemical modification. TGA revealed that the surface-functionalized CNCs present higher thermal stability than pure CNCs. The confocal microscopy, zeta potential, and DLS results were consistent with the behavior of cellulose nanocrystals decorated by a shell of PNIPAM chains. The fluids with a small amount of modified CNCs presented a much lower volume of filtrate after high-temperature and high-pressure (HTHP) filtration tests than the corresponding standard fluid, indicating the applicability of the environmentally friendly particles for olefin-based drilling fluids.     

Catalytic Cu(II)–polymer complexes as recyclable catalysts for the synthesis of poly(2,6-dimethyl-1,4-phenylene oxide)s in water

Water-soluble polymers comprising itaconic amide acid with acrylic acid or acrylamide, which contain carboxylic acid and amide groups capable of coordinating to the copper catalyst, were synthesized by radical polymerization using an azobisisobutyronitrile initiator. These polymers were used as polymer ligands to prepare copper complexes, which were subsequently analyzed by UV–Vis spectroscopy. The complexes were then used as catalysts for the oxidative polymerization of 2,6-dimethylphenol (DMP) to synthesize poly(2,6-dimethyl-1,4-phenylene oxide) (PPO) under oxygen and in the presence of a surfactant in alkaline water. The polymerization conditions were optimized by varying the amounts of polymer ligands and copper precursors, the concentrations of surfactant and hydrogen chloride, and the temperature, resulting in PPO with a maximum yield of 93%, a number-average molecular weight (M _n) of 3700, and a molecular weight distribution (M _w/M _n) of 2.12. This yield is higher than that previously achieved using arginine ligand in water (72%). Furthermore, the optimum conditions were applied in the copolymerization of DMP and 2-allyl-6-methylphenol to obtain a thermally crosslinkable copolymer in 95% yield (M _n = 3000, M _w/M _n = 2.5). In addition, the catalyst complex of the copper–polymer ligand was recovered and reused after the polymerization of DMP. The catalyst maintained its activity even after being recycled five times, without the addition of copper precursor or polymer ligand, thereby demonstrating an environmentally friendly process wherein environmental emissions and production costs can be substantially reduced.