Maleic Acid Copolymer Research Articles

Functional reconstitution of human equilibrative nucleoside transporter-1 into styrene maleic acid co-polymer lipid particles

The human equilibrative nucleoside transporter-1 (hENT1) is important for the entry of anti-cancer and anti-viral nucleoside analog therapeutics into the cell, and thus for their efficacy. Understanding of hENT1 structure-function relationship could assist with development of nucleoside analogs with better cellular uptake properties. However, structural and biophysical studies of hENT1 remain challenging as the hydrophobic nature of the protein leads to complete aggregation upon detergent-based membrane isolation. Here we report detergent-free reconstitution of the hENT1 transporter into styrene maleic acid co-polymer lipid particles (SMALPs) that form a native lipid disc. SMALP-purified hENT1, expressed in Sf9 insect cells binds a variety of ligands with a similar affinity as the protein in native membrane, and exhibits increased thermal stability compared to detergent-solubilized hENT1. hENT1-SMALPs purified using FLAG affinity M2 resin yielded ~0.4mg of active and homogenous protein per liter of culture as demonstrated by ligand binding, size-exclusion chromatography and SDS-PAGE analyses. Electrospray ionization mass spectrometry (ESI-MS) analysis showed that each hENT1 lipid disc contains 16 phosphatidylcholine (PC) and 2 phosphatidylethanolamine (PE) lipid molecules. Polyunsaturated lipids are specifically excluded from the hENT1 lipid discs, possibly reflecting a functional requirement for a dynamic lipid environment. Our work demonstrates that human nucleoside transporters can be extracted and purified without removal from their native lipid environment, opening up a wide range of possibilities for their biophysical and structural studies.

Crystallogenesis of Membrane Proteins Mediated by Polymer-Bounded Lipid Nanodiscs

Summary For some membrane proteins, detergent-mediated solubilization compromises protein stability and functionality, often impairing biophysical and structural analyses. Hence, membrane-protein structure determination is a continuing bottleneck in the field of protein crystallography. Here, as an alternative to approaches mediated by conventional detergents, we report the crystallogenesis of a recombinantly produced membrane protein that never left a lipid bilayer environment. We used styrene–maleic acid (SMA) copolymers to solubilize lipid-embedded proteins into SMA nanodiscs, purified these discs by affinity and size-exclusion chromatography, and transferred proteins into the lipidic cubic phase (LCP) for in meso crystallization. The 2.0-A structure of an α-helical seven-transmembrane microbial rhodopsin thus obtained is of high quality and virtually identical to the 2.2-A structure obtained from traditional detergent-based purification and subsequent LCP crystallization.

The effect of a triclosan/copolymer/fluoride toothpaste on plaque formation, gingivitis, and dentin hypersensitivity: A single-blinded randomized clinical study.

The daily removal of supragingival dental plaque is a key factor in the prevention of gingivitis. The aim of the study was to compare the gingival health benefits of a triclosan/copolymer/fluoride toothpaste (Colgate Total, a fluoride toothpaste containing an antiseptic) to a commercially available toothpaste containing 0.243% sodium fluoride in a silica base (Colgate Herbal, a conventional fluoride toothpaste with herbal extracts). A total of 50 patients with gingivitis and at least one sensitive tooth were included. The subjects were randomly stratified into two groups: Colgate Total toothpaste, and Colgate Herbal toothpaste. After a 4-week pre-experimental phase, baseline Plaque Index (Quigley-Hein Index) (PI), Gingival Index (GI), Gingival Bleeding Index (GBI), and Visual Analog Scale (VAS) were assessed. The PI, GI, GBI, and VAS were reexamined at weeks 4, 12, and 24 after the baseline. Fifty subjects complied with the protocol and completed the study. The conventional fluoride toothpaste with herbal extracts group and the fluoride toothpaste containing an antiseptic group exhibited significant reductions in PI, GI, GBI, and VAS over time. The amount of reduction after 6 months of the treatment was higher in the Total group compared to Herbal group (1.82 vs 1.39, P = .015 for PI; 0.67 vs 0.37, P < .005 for GI; and 56.64% vs 34.26%, P < .005 for GBI). No significant difference was seen for VAS. Twice daily brushing with a toothpaste containing 0.3% triclosan and polyvinyl methyl ether and maleic acid copolymer provides a more effective level of plaque control and gingival health with no effect on decreasing dentin hypersensitivity compared to conventional fluoride toothpaste. Toothpastes containing triclosan/copolymer, in addition to fluoride, result in a higher reduction in plaque, gingival inflammation, and gingival bleeding when compared with fluoride toothpastes without triclosan/copolymer.

Determination of the effectiveness of new green scale inhibitors for reverse osmosis

The effectiveness of a number the test samples of new antiscalants (lightly crosslinked polymers of acrylic and methacrylic acids and crosslinking agents based on allyl ethers and other compounds, and a copolymer of maleic anhydride and methacrylic acid) has been experimentally compared with that of traditionally used scale inhibitors based on hydroxyethylidene diphosphonic acid, nitrilotriphosphonic acid, and the commercial inhibitor Aminat-K. The results of the experimental study of the kinetics of formation and buildup of the crystalline deposit of calcium carbonate in the presence and the absence of scale inhibitors depending on the composition of feedwater and the yield of a filtrate are reported. The experiments have been performed with the use of feedwater from the Moscow water-supply line. Equations for determining the rates of formation of calcium carbonate deposits in membrane devices in the presence of an inhibitor depending on the chemical composition of feedwater and the filtrate yield have been derived; these equations make it possible to recommend a work-cycle time before chemical cleaning operations.

Isolation of yeast complex IV in native lipid nanodiscs

We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11nm×14nm. Based on this size we estimated that each CytcO was surrounded by ~100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane. Even though CytcO forms a supercomplex with cytochrome bc1 in the mitochondrial membrane, cyt. bc1 was not found in the native nanodiscs. Yet, the loosely-bound Respiratory SuperComplex factors were found to associate with the isolated CytcO. The native nanodiscs displayed an O2-reduction activity of ~130 electrons CytcO−1s−1 and the kinetics of the reaction of the fully reduced CytcO with O2 was essentially the same as that observed with CytcO in mitochondrial membranes. The kinetics of CO-ligand binding to the CytcO catalytic site was similar in the native nanodiscs and the mitochondrial membranes. We also found that excess SMA reversibly inhibited the catalytic activity of the mitochondrial CytcO, presumably by interfering with cyt. c binding. These data point to the importance of removing excess SMA after extraction of the membrane protein. Taken together, our data shows the high potential of using SMA-extracted CytcO for functional and structural studies.

Membrane proteins: is the future disc shaped?

The use of styrene maleic acid lipid particles (SMALPs) for the purification of membrane proteins (MPs) is a rapidly developing technology. The amphiphilic copolymer of styrene and maleic acid (SMA) disrupts biological membranes and can extract membrane proteins in nanodiscs of approximately 10nm diameter. These discs contain SMA, protein and membrane lipids. There is evidence that MPs in SMALPs retain their native structures and functions, in some cases with enhanced thermal stability. In addition, the method is compatible with biological buffers and a wide variety of biophysical and structural analysis techniques. The use of SMALPs to solubilize and stabilize MPs offers a new approach in our attempts to understand, and influence, the structure and function of MPs and biological membranes. In this review, we critically assess progress with this method, address some of the associated technical challenges, and discuss opportunities for exploiting SMA and SMALPs to expand our understanding of MP biology.

Styrene maleic acid copolymers mediate selective release of protein targets from the periplasm of E. coli

Styrene maleic acid copolymers mediate selective release of protein targets from the periplasm of E. coli

A method for detergent-free isolation of membrane proteins in their local lipid environment.

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

RETRACTED: pH-Responsive Polymer Conjugate of Pirarubicin With Styrene Maleic Acid Copolymer as a Potential Therapeutic for Ovarian Cancer

Previous studies indicated the potential of styrene maleic acid copolymer (SMA)-conjugated pirarubicin (4'-O-tetrahydropyranyldoxorubicin [THP]) for targeted anticancer therapy based on the enhanced permeability and retention effect. In this study, to achieve further improved therapeutic efficacy, a pH-responsive SMA-conjugated THP-containing hydrazone bond (SMA-hyd-THP) was synthesized and evaluated invitro and exvivo using human ovarian cancer cells and tissues. SMA-hyd-THP showed good water solubility, forming micelles with a mean particle size of 48.0 nm, which is applicable for enhanced permeability and retention-based tumor accumulation. The THP loading in this preparation was 15% (wt/wt), and release rate of free THP from SMA-hyd-THP at physiological pH (7.4) was approximately 10% in 72h. However, it increased rapidly at pH 6.5 (42%) and 5.5 (83%), which indicates that tumor environment of weak acidic condition (pH 6.5-6.9) is favorable for release of THP. This notion was partly proved by incubating SMA-hyd-THP with tumor tissues from ovarian cancer patients. In addition, release of THP was not affected by serum, suggesting that SMA-hyd-THP is relatively stable in circulation. Finally, SMA-hyd-THP showed much increased cytotoxicity against various ovarian cancer cells at acidic tumor pH (6.5). These findings may provide an option for targeted therapy against ovarian cancer.

SMA-SH: Modified Styrene-Maleic Acid Copolymer for Functionalization of Lipid Nanodiscs.

Challenges in purification and subsequent functionalization of membrane proteins often complicate their biochemical and biophysical characterization. Purification of membrane proteins generally involves replacing the lipids surrounding the protein with detergent molecules, which can affect protein structure and function. Recently, it was shown that styrene-maleic acid copolymers (SMA) can dissolve integral membrane proteins from biological membranes into nanosized discs. Within these nanoparticles, proteins are embedded in a patch of their native lipid bilayer that is stabilized in solution by the amphipathic polymer that wraps the disc like a bracelet. This approach for detergent-free purification of membrane proteins has the potential to greatly simplify purification but does not facilitate conjugation of functional compounds to the membrane proteins. Often, such functionalization involves laborious preparation of protein variants and optimization of labeling procedures to ensure only minimal perturbation of the protein. Here, we present a strategy that circumvents several of these complications through modifying SMA by grafting the polymer with cysteamine. The reaction results in SMA that has solvent-exposed sulfhydrils (SMA-SH) and allows tuning of the coverage with SH groups. Size exclusion chromatography, dynamic light scattering, and transmission electron microscopy demonstrate that SMA-SH dissolves lipid bilayer membranes into lipid nanodiscs, just like SMA. In addition, we demonstrate that, just like SMA, SMA-SH solubilizes proteoliposomes into protein-loaded nanodiscs. We covalently modify SMA-SH-lipid nanodiscs using thiol-reactive derivatives of Alexa Fluor 488 and biotin. Thus, SMA-SH promises to simultaneously tackle challenges in purification and functionalization of membrane proteins.

Influence of lipid bilayer properties on nanodisc formation mediated by styrene/maleic acid copolymers.

Copolymers of styrene and maleic acid (SMA) have gained great attention as alternatives to conventional detergents, as they offer decisive advantages for studying membrane proteins and lipids in vitro. These polymers self-insert into artificial and biological membranes and, at sufficiently high concentrations, solubilise them into disc-shaped nanostructures containing a lipid bilayer core surrounded by a polymer belt. We have used (31)P nuclear magnetic resonance spectroscopy and dynamic light scattering to systematically study the solubilisation of vesicles composed of saturated or unsaturated phospholipids by an SMA copolymer with a 3 : 1 styrene/maleic acid molar ratio at different temperatures. Solubilisation was thermodynamically rationalised in terms of a three-stage model that treats various lipid/polymer aggregates as pseudophases. The solubilising capacity of SMA(3 : 1) towards a saturated lipid is higher in the gel than in the liquid-crystalline state of the membrane even though solubilisation is slower. Although the solubilisation of mixed fluid membranes is non-selective, the presence of a non-bilayer phospholipid lowers the threshold at which the membrane becomes saturated with SMA(3 : 1) but raises the polymer concentration required for complete solubilisation. Both of these trends can be explained by considering the vesicle-to-nanodisc transfer free energies of the lipid and the polymer. On the basis of the phase diagrams thus obtained, re-association of polymer-solubilised lipids with vesicles is possible under mild conditions, which has implications for the reconstitution of proteins and lipids from nanodiscs into vesicular membranes. Finally, the phase diagrams provide evidence for the absence of free SMA(3 : 1) in vesicular lipid suspensions.

A Comparative Performance Evaluation of Some Novel “Green” and Traditional Antiscalants in Calcium Sulfate Scaling

A relative ability of industrial samples of four phosphorus-free polymers (polyaspartate (PASP); polyepoxysuccinate (PESA); polyacrylic acid sodium salt (PAAS); copolymer of maleic and acrylic acid (MA-AA)) and of three phosphonates (aminotris(methylenephosphonic acid), ATMP; 1-hydroxyethane-1,1-bis(phosphonic acid), HEDP; phosphonobutane-1,2,4-tricarboxylic acid, PBTC) to inhibit calcium sulfate precipitation is studied following the NACE Standard along with dynamic light scattering (DLS), scanning electron microscopy (SEM), and X-ray diffraction (XRD) technique. For the 0.5 mg·dm−3dosage, the following efficiency ranking was found:MA-AA~ATMP&gt;PESA (400–1500 Da)&gt;PASP (1000–5000 Da)≫PAAS (3000–5000 Da)~PBTC~HEDP. The isolated crystals are identified as gypsum. SEM images for PESA, PASP, PAAS, and HEDP and for a blank sample indicated the needle-like crystal morphology. Surprisingly, the least effective reagent PBTC revealed quite a different behavior, changing the morphology of gypsum crystals to an irregular shape. The DLS experiments exhibited a formation of 300 to 700 nm diameter particles with negativeζ-potential around −2 mV for all reagents.Although suchζ-potential values are not capable of providing colloidal stability, all three phosphonates demonstrate significant gypsum particles stabilization relative to a blank experiment.

The styrene-maleic acid copolymer: a versatile tool in membrane research.

A new and promising tool in membrane research is the detergent-free solubilization of membrane proteins by styrene–maleic acid copolymers (SMAs). These amphipathic molecules are able to solubilize lipid bilayers in the form of nanodiscs that are bounded by the polymer. Thus, membrane proteins can be directly extracted from cells in a water-soluble form while conserving a patch of native membrane around them. In this review article, we briefly discuss current methods of membrane protein solubilization and stabilization. We then zoom in on SMAs, describe their physico-chemical properties, and discuss their membrane-solubilizing effect. This is followed by an overview of studies in which SMA has been used to isolate and investigate membrane proteins. Finally, potential future applications of the methodology are discussed for structural and functional studies on membrane proteins in a near-native environment and for characterizing protein–lipid and protein–protein interactions.

Behavior of thermo- and pH-responsive copolymer of N-isopropylacrylamide and maleic acid in aqueous solutions

ABSTRACTA thermo- and pH-responsive copolymer of N-isopropylacrylamide with maleic acid was studied using light scattering and turbidimetry methods. Aqueous solutions with pH values from 1.8 to 10.9 and in the concentration range from 0.001 to 0.015 g/cm3 were investigated. At all pH values and concentrations, phase separation was observed at temperatures T > 33°C. The temperatures of the start and the width of the phase separation interval increased with decrease of copolymer concentration and increase in pH. The redistribution of scattering entities, namely, macromolecular unimers, micelle-like structures, and loose aggregates, and growth of aggregate hydrodynamic radius took place during heating.

Determination of amikacin by Rayleigh scattering method based on the covalent bonding of the analyte with a water soluble polymer

A method for the determination of aminoglycoside antibiotics based on the covalent bonding of the analyte with a water-soluble polymer by the Rayleigh scattering method (λex = λem) is proposed. Determination of amikacin (as a model analyte) is carried out by the carbodiimide crosslinking technique, which forms amide bonds between the copolymer of 4-styrenesulfonic acid and maleic acid and the primary amine groups of amikacin. The conditions of the reaction are selected to obtain maximum scattering intensity of the product: reaction time 15 min, pH 7.0, ionic strength 0.02, and measuring of scattering intensity at 362 nm. The determination of amikacin is not influenced by the following (mol/mol ratios): Al(III), Co(II), Cu(II), Fe(III), Ni(II), Zn(II)—300; Cl– and NO3-—250; Ca(II) and Mg(II)—50; HCO3-—20; it is affected by nitrogen compounds at 20–50-fold ratios and proteins at a 2.5-fold ratio. The calibration plot is constructed over the range of 0.2–35 mg/L (r = 0.993); LOD (3s-criterium) is 0.08 mg/L (n = 10, P = 0.95). Analyses are performed on drug powder for injection and gel.

Behaviorial Features of Aqueous Solutions of Thermoresponsive and pH-Sensitive Polymers with complicated architectures

Research on self-organization and aggregation in solutions of thermoresponsive and pH-sensitive polymers with complicated architectures such as star-shaped polyoxazolines and grafted copolymers with a polyimide spine and polydimethylaminoethylmethacrylate side chains was reviewed. A statistical N-isopropylacrylamide—maleic-acid copolymer was studied for comparison. Hydrophobic blocks in the macromolecules reduced the phase-stratification temperature and changed the set of scattering species by forming monomolecular micelles. An increased intramolecular density led to slower aggregate formation in solutions of polymers with complicated architectures.

Synthesis and Investigation of Double Stimuli-Responsive Behavior of N-Isopropylacrylamide and Maleic Acid Copolymer in Solutions

Poly(N-isopropylacrylamide-co-maleic acid) [poly(NIPAAm-co-MA)] linear copolymer with comonomer molar ratio NIPAAm:MA = 94:6 was synthesized by free-radical copolymerization. The molar mass, MsD = 28,000 Da, of poly(NIPAAm-co-MA) was determined using hydrodynamic methods. The self-assembly of poly(NIPAAm-co-MA) in aqueous solution at a concentration of 0.015 g cm−3 within the pH interval from 1.8 to 10.6 and the temperature interval from 22 to 60°C was investigated by static and dynamic light scattering. The copolymer showed a double temperature and pH responsiveness. Three types of particles, namely, macromolecules (or unimers), micellar-like structures, and loose aggregates existed in the poly(NIPAAm-co-MA) aqueous solutions. The fraction of dissolved entities and the hydrodynamic radii of the micellar-like structures and loose aggregates depended considerably on temperature and pH. The temperature of the phase separation and the width of the phase separation interval increased with pH. An influence of pH on kinetic processes in poly(NIPAAm-co-MA) solutions was observed.

Styrene maleic acid copolymer–pirarubicin induces tumor-selective oxidative stress and decreases tumor hypoxia as possible treatment of colorectal cancer liver metastases

Pirarubicin, a derivative of doxorubicin, induces tumor destruction via the production of reactive oxygen species (ROS) but is associated with cardiotoxicity. As a macromolecule (conjugated to styrene-maleic acid [SMA]), SMA-pirarubicin is selective to tumors resulting in improved survival with decreased systemic toxicity. Tumor destruction is, however incomplete, and resistant cells at the periphery of the tumor contribute to recurrence. Tumor hypoxia is a major factor in tumor resistance. Understanding the effect of oxidative stress induced by SMA-pirarubicin on the tumor microenvironment may be key to overcoming resistance. This study investigated the pattern of ROS production and tumor hypoxia after treatment with SMA-pirarubicin in a murine model of colorectal liver metastases. Liver metastases were induced in male, CBA mice using a murine-derived colon cancer cell line. SMA-pirarubicin (maximum tolerated dose, 100 mg/kg) or pirarubicin, (maximum tolerated dose, 10 mg/kg) were administered intravenously 14 days after tumor induction. Systemic oxidative stress in serum, liver, and cardiac tissue was quantified using the thiobarbituric acid reactive substances assay. Flow cytometry and fluorescence microscopy were used to assess ROS production for 48 hours after treatment. Tumor hypoxia was quantified using immunohistochemistry for pimonidazole adducts. SMA-pirarubicin (100 mg/kg) induced ROS exclusively in tumors with minimal levels in serum and cardiac tissue. ROS levels were induced in a time-dependent and dose-dependent manner optimal between 4 and 24 hours after drug administration. Although tumor hypoxia was decreased overall, residual tumor cells adjacent to patent vessels were hypoxic. This study provides insight into the tumor microenvironment after chemotherapy. SMA-pirarubicin inhibits the growth of colorectal liver metastases by inducing ROS, which seems to be largely tumor selective. The temporal pattern of ROS production can be used to improve future dosing regimens. Furthermore, the observation that residual tumor cells are hypoxic clarifies the need for a multimodal approach with agents that can alter the hypoxic state to effect complete tumor destruction.

G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent.

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([3H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

INVESTIGATION OF ANTIMICROBIAL PROPERTIES OF SILVER NANOPARTICLES STABILIZED BY MALEIC ACID COPOLYMERS

Silver nanoparticles have been prepared by the reduction of the corresponding metal salt with NaBH 4 in the presence of appropriate dispersing agents, namely, copolymers of maleic acid with N-vinylpyrrolidone, ethylene and styrene or their amphyphi- lic derivative containing octadecylamide ligands. The silver colloids identified by optical absorption spectroscopy were synthesized when the copolymer concentrations were lower than their critical aggregation concentrations and copolymer/Ag+ molar ratio no greater than a unit. According to the data of transmission electron microscopy, the obtained Ag nanoparticles were spherical-shaped particles 1—2 nm in size. Colloidal solutions of the prepared nanosilver showed a strong inhibiting antifungal activity against a fungal plant pathogen Fusarium oxysporum and also Gram-negative, Gram-positive bacteria and yeast. The silver nanoparticles stabilized with more hydrophilic compounds from among the investigated copolymers demonstrated the higher antibacterial and antifungal activity.