Chromatographic adsorption of serum albumin and antibody proteins in cryogels with benzyl-quaternary amine ligands.

The preparation and characterization of mixed-mode adsorbents for a typical separation purpose are of great importance in bioseparation areas. In this work, we prepared a new monolithic cryogel with a combination of ion-exchange and hydrophobic functions by employing benzyl-quaternary amine groups. The fundamental cryogel properties, protein equilibrium adsorption isotherm and chromatographic adsorption in the cryogel were measured experimentally. The results showed that, by using bovine serum album as the model protein, the dual functional cryogel has protein binding capability even in salt solution and the buffer with pH close or below the protein isoelectric point due to both the electrostatic and hydrophobic interactions. A capillary-based adsorption model was developed, which provided satisfied insights of the microstructure, axial dispersion, mass transfer as well as protein adsorption characteristics within the cryogel bed. The chromatographic isolation of bioactive proteins from rabbit blood serum was carried out by the cryogel. Immunoglobulin G antibody with a purity of 98.2% and albumin with a purity of 96.8% were obtained, indicating that the cryogel could be an interesting and promising adsorbent in bioseparation areas.

Poly(hydroxyethyl methacrylate)-based composite cryogel with embedded macroporous cellulose beads for the separation of human serum immunoglobulin and albumin.

A novel super-macroporous monolithic composite cryogel was prepared by embedding macroporous cellulose beads into poly(hydroxyethyl methacrylate) cryogel. The cellulose beads were fabricated by using a microchannel liquid-flow focusing and cryopolymerization method, while the composite cryogel was prepared by cryogenic radical polymerization of the hydroxyethyl methacrylate monomer with poly(ethylene glycol) diacrylate as cross-linker together with the cellulose beads. After graft polymerization with (vinylbenzyl)trimethylammonium chloride, the composite cryogel was applied to separate immunoglobulin-G and albumin from human serum. Immunoglobulin-G with a mean purity of 83.2% and albumin with a purity of 98% were obtained, indicating the composite cryogel as a promising chromatographic medium in bioseparation for the isolation of important bioactive proteins like immunoglobulins and albumins.

Isolation of immunoglobulin G from bovine milk whey by poly(hydroxyethyl methacrylate)-based anion-exchange cryogel.

Bovine milk whey contains several bioactive proteins such as α-lactalbumin, ß-lactoglobulin, and immunoglobulin G (IgG). Chromatographic separation of these proteins has received much attention in the past few years. In this work, we provide a chromatographic method for the efficient isolation of IgG from bovine milk whey using a poly(2-hydroxyethyl methacrylate)-based anion-exchange cryogel. The monolithic cryogel was prepared by grafting 2-(dimethylamino) ethyl methacrylate onto the poly(2-hydroxyethyl methacrylate)-based cryogel matrix and then employed to separate IgG under various buffer pH and salt elution conditions. The results showed that the buffer pH and the salt concentration in the step elution have remarkable influences on the purity of IgG, while the IgG recovery depended mainly on the loading volume of whey for a given cryogel bed. High purity IgG (more than 95%) was obtained using the phosphate buffer with pH of 5.8 as the running buffer and the salt solution in as the elution liquid. With suitable loading volume of whey, the maximum IgG recovery of about 94% was observed. The present separation method is thus a potential choice for the isolation of high-purity IgG from bovine milk whey.

Cationic salt-responsive bottle-brush polymers.

A class of cationic bottle-brush polymers that show ionic strength-dependent stimuli responsiveness is prepared. Brush polymers with norbornene as backbone and quaternary ammonium (QA)-containing polycaprolactone copolymers as side chains are synthesized by a combination of ring-opening metathesis polymerization, ring-opening polymerization, and click reaction. In water with low ionic strength, brush polymers are soluble due to the strong electrostatic repulsion between cationic QA groups. As the addition of salt to increase ionic strength, single brush polymers undergo a transition from extended conformation to collapsed state and finally become insoluble in solution due to the screening effect of salts that yield the once-dominant electrostatic interactions among QA species to hydrophobic-hydrophobic interactions.

Synthesis and drug delivery of novel amphiphilic block copolymers containing hydrophobic dehydroabietic moiety.

Well-defined amphiphilic poly(ethylene glycol) and poly(dehydroabietic ethyl methacrylate) block copolymers (PEG-b-PDAEMA) were prepared by atom transfer radical polymerization. The methacrylate block contains a characteristic hydrophobic, biocompatible and economical dehydroabietic moiety. PEG-b-PDAEMA block copolymer micellar nanoparticles loaded with piperlongumine (PLGM) were successfully prepared by a nanoprecipitation method. In vitro and in vivo behaviors of these nanoparticles were thoroughly examined by a set of characterization techniques. Confocal laser scanning microscopy study revealed that these nanoparticles could be well taken up by cancer cells. In vivo near-infrared fluorescence imaging showed that the PLGM-loaded nanoparticles effectively targeted the tumor site by the enhanced permeability and retention (EPR) effect in H22 tumor-bearing mice. The in vivo antitumor examination found that PLGM-loaded nanoparticles exhibited superior efficacy in impeding the tumor growth compared to the commercially available Taxol® and free PLGM formulation. The changes in body weights and blood biochemical profiles were also compared to investigate the safety of PLGM and PEG-b-PDAEMA nanoparticle drug delivery system.

Building triangular nanoprisms from the bottom-up: a polyelectrolyte micellar approach.

Charge-regulated synthesis of triangular prisms in aqueous solution using self-assembled polyelectrolyte micelles as templates is described in detail. Micelles formed from amphiphilic polystyrene-block-sulfonated poly(1,3-cyclohexadiene) (PS-b-sPCHD) serve as templates to direct the formation of novel triangular prisms of CuCl2 single crystals. We demonstrate that the edge lengths of these triangular prisms can be easily tailored at room temperature from the nanoscale to the mesoscale by simply adjusting the ratio of charged micelles to protons in the solution. This approach can be extended to the preparation of different ordered crystal structures with a precision hard to achieve via other approaches.

[Chromatographic separation of plasmid DNA by anion-exchange cryogel].

Plasmid DNA (pDNA) is used as an important vector for gene therapy, and its wide application is restricted by the purity and yield. To obtain high-purity pDNA, a chromatographic method based on anion-exchange supermacroporous cryogel was explored. The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method. The plasmid was transferred into Escherichia coli DH5alpha, cultivated, harvested and lysed. The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock, which was loaded into the anion-exchange cryogel bed for chromatographic separation. By optimizing the pH of running buffer and the elution conditions, high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6. Compared to the traditional methods for purification of pDNA, animal source enzymes and toxic reagents were not involved in the present separation process, ensuring the safety of both the purification operations and the obtained pDNA.

Microchannel liquid-flow focusing and cryo-polymerization preparation of supermacroporous cryogel beads for bioseparation.

Polymeric cryogels are sponge-like materials with supermacroporous structure, allowing them to be of interest as new chromatographic supports, cell scaffolds and drug carriers in biological and biomedical areas. The matrices of cryogels are always prepared in the form of monoliths by cryo-polymerization under frozen conditions. However, there are limited investigations on the production of cryogels in the form of adsorbent beads suitable for bioseparation. In this work, we provide a new approach by combining the microchannel liquid-flow focusing with cryo-polymerization for the preparation of polyacrylamide-based supermacroporous cryogel beads with a narrow particle size distribution. The present method was achieved by introducing the aqueous phase solution containing monomer, cross-linker and redox initiators, and the water-immiscible organic oil phase containing surfactant simultaneously into a microchannel with a cross-shaped junction, where the aqueous drops with uniform sizes were generated by the liquid shearing and the segmentation due to the steady flow focusing of the immiscible phase streams. These liquid drops were in situ suspended into the freezing bulk oil phase for cryo-polymerization and the cryogel matrix beads were obtained by thawing after the achievement of polymerization. By grafting the polymer chains containing sulfo binding groups onto these matrix beads, the cation-exchange cryogel beads for protein separation were produced. The results showed that at the aqueous phase velocities from 0.5 to 2.0 cm/s and the total velocities of the water-immiscible phase from 2.0 to 6.0 cm/s, the obtained cryogel beads by the present method have narrow size distributions with most of the bead diameters in the range from 800 to 1500 µm with supermacropores in sizes of about 3-50 µm. These beads also have high porosities with the averaged maximum porosity of 96.9% and the mean effective porosity of 86.2%, which are close to those of the polyacrylamide-based cryogel monoliths. The packed bed using the cryogel beads with mean diameter of 1248 µm, as an example, has reasonable and acceptable liquid dispersion, but high water permeability (4.29 × 10⁻¹° m²) and high bed voidage (90.2%) owing to the supermacropores within the beads, enhanced the rapid binding and separation of protein from the feedstock even at high flow velocities. The purity of the obtained lysozyme from chicken egg white by one-step chromatography using the packed bed was in the range of about 78-92% at the flow velocities of 0.5-15 cm/min, indicating that the present cryogel beads could be an effective chromatographic adsorbent for primary bioseparation.

Robust antimicrobial compounds and polymers derived from natural resin acids.

We report novel robust resin acid-derived antimicrobial agents that exhibit excellent antimicrobial activities against a broad spectrum of bacteria (6 Gram-positive and 7 Gram-negative) with selective lysis of microbial membranes over mammalian membranes. Our results indicate that hydrophobicity and unique structures of resin acids can be determining factors in dictating the antimicrobial activity.

Degradable rosin-ester-caprolactone graft copolymers.

We have carried out the synthesis of side-chain rosin-ester-structured poly(ε-caprolactone) (PCL) through a combination of ring-opening polymerization and click chemistry. Rosin structures are shown to be effectively incorporated into each repeat unit of caprolactone. This simple and versatile methodology does not require sophisticated purification of raw renewable biomass from nature. The rosin properties have been successfully imparted to the PCL polymers. The bulky hydrophenanthrene group of rosin increases the glass-transition temperature of PCL by >100 °C, whereas the hydrocarbon nature of rosin structures provides PCL excellent hydrophobicity with contact angle very similar to polystyrene and very low water uptake. The rosin-containing PCL graft copolymers exhibit full degradability and good biocompatibility. This study illustrates a general strategy to prepare a new class of renewable hydrocarbon-rich degradable biopolymers.

Isolation of ATP from a yeast fermentation broth using a cryogel column at high flow velocities.

This communication presents an effective method for isolating adenosine triphosphate (ATP) from a yeast fermentation broth using an anion-exchange supermacroporous cryogel column at high flow velocities. The breakthrough and elution behaviors of pure ATP in the cryogel bed were investigated at flow velocities of 2, 5, and 10 cm/min and the ATP binding capacities were determined. Then the ATP-containing yeast fermentation broth was employed as the test feedstock and various chromatographic runs were conducted to isolate ATP by the cryogel at different high flow velocities. The ATP samples obtained were analyzed quantitatively by HPLC. The results showed that even at a flow velocity of 5 or 10 cm/min, a product purity of 97.4 or 98.0% can be achieved, illustrating the potential of the present method for separation of high-purity ATP directly from fermentation feedstock at high flow velocities.

Chromatographic separation of cytidine triphosphate from fermentation broth of yeast using anion-exchange cryogel.

A novel separation method was developed to isolate directly cytidine triphosphate (CTP) from fermentation broth of yeast using anion-exchange supermacroporous cryogel. The anion-exchange cryogel with tertiary amine groups was prepared by graft polymerization. The breakthrough characteristics and elution performance of pure CTP in the cryogel bed were investigated experimentally and the CTP binding capacity was determined. Then the separation experiments of CTP from crude fermentation broth of yeast using the cryogel column were carried out using deionized water and 0.01 M HCl as washing buffer, respectively. The chromatographic behavior was monitored and analyzed. The purity and concentration of the obtained CTP in these processes were determined quantitatively by HPLC. The maximal purity of CTP obtained at the condition of 0.01 M HCl as washing buffer and 0.5 M NaCl in 0.01 M HCl as elution buffer reached 93%.

One-step isolation of adenosine triphosphate from crude fermentation broth of Saccharomyces cerevisiae by anion-exchange chromatography using supermacroporous cryogel.

Adenosine triphosphate (ATP) is an important high-energy compound widely used in biological and therapeutic fields. It can be produced by phosphorylation of adenosine monophosphate (AMP) with microbial cells in industrial scale and the effective isolation of ATP from microbial fermentation broth is a challenging work. In this work, we develop a novel one-step method to directly separate ATP from fermentation broth of Saccharomyces cerevisiae by anion-exchange chromatography using supermacroporous cryogel. The cryogel bed with tertiary amine groups was prepared by grafting N,N-dimethylaminoethyl methacrylate (DMAEMA) monomer chains onto the matrix of a polyacrylamide-based cryogel in a glass column and its properties of liquid dispersion, water permeability, porosity as well as the ligand density were measured. Chromatographic separation of ATP from the fermentation broth by the cryogel was carried out using deionised water and 0.01 M HCl as running buffer, respectively. The breakthrough characteristics and elution performance in the cryogel bed were revealed and analyzed. The purities of the obtained ATP were analyzed quantitatively by high performance liquid chromatography (HPLC). The maximal purity of ATP by the one-step separation method was 95.5% using 0.01 M HCl as running buffer in this work. The corresponding chromatographic behaviors were investigated and analyzed.

In-situ graft-polymerization preparation of cation-exchange supermacroporous cryogel with sulfo groups in glass columns.

Graft polymerization of monomer chains with expected functional groups onto the matrix pore surfaces by initiator is an effective approach for introducing ion-exchange groups to cryogel matrix to get anion- or cation-exchange supermacroporous cryogels. In this work, a novel cation-exchange cryogel with sulfo binding groups was prepared by grafting of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) onto polyacrylamide-based cryogels in glass columns. The grafting polymerization was achieved in an in-situ manner which was performed by pumping the initiator and the reactive solution of graft monomer with sulfo binding groups directly through a cryogel bed pre-produced in a glass column under frozen condition. The axial liquid dispersion characteristics within the monolithic cryogel beds before and after the in-situ polymerization were compared by measuring residence time distributions (RTDs) at various liquid flow rates using tracer pulse-response method. Microstructure morphology of pores within cryogels was analyzed by scanning electron microscopy (SEM). Chromatography of lysozyme was carried out to reveal the protein breakthrough and elution characteristics in the obtained cryogel beds.

Characterization of a novel continuous supermacroporous monolithic cryogel embedded with nanoparticles for protein chromatography.

A novel continuous supermacroporous monolithic cryogel embedded with nanometer-size particles was prepared by the radical cryogenic co-polymerization of acrylamide (AAm), N,N'-methylene-bis-acrylamide (MBAAm), allyl glycidyl ether (AGE) and the dispersed surfactant-stabilized Fe3O4 nanoparticles under the freezing-temperature variation condition in a glass column. This special separation matrix has interconnected supermacropores with pore size of 10-50 microm, which permit the free-passage of microbial cells or cell debris in the culture fluids and then is interest in downstream processes. The axial liquid dispersion coefficients of the new continuous supermacroporous monolithic bed at different liquid flow rates were obtained by measuring residence time distributions (RTDs) using tracer pulse-response method. The experimental results showed that the axial liquid dispersion within the bed was weak in a wide water flow rate of 0.5-15 cm/min. The axial dispersion coefficient was found to be increased exponentially with the increase of liquid flow rate. Chromatographic process of bovine serum albumin (BSA) in the cryogel monolithic bed was carried out to reveal the protein breakthrough and elution characteristics. Compared with other reported cryogel beds in literature, the protein adsorption capacity of the present cryogel bed was improved due to the embedded nano-sized solid adsorbents in the gel matrix. Microstructure morphology of the embedded nanoparticles in the cryogel and the gel matrix structure were also analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in this paper.