Development of an aspartic acid-based cross-linking monomer for improved bioseparations

Improved specificity and binding affinity by molecularly imprinted polymers is possible by development of novel functional materials. Furthermore, increasing the cross-link density of imprinted polymers by using cross-linking functional groups was anticipated to improve polymer molecular recognition. A novel cross-linking monomer derived from an L-aspartic acid precursor was synthesized and employed in molecularly imprinted polymers to mimic more closely the scaffolding of proteins, and thus provide more protein-like selectivity. Chromatographic results revealed a more than 7-fold improvement in polymers imprinted using the new monomer versus a traditionally formulated polymer imprinted with methacrylic acid as the functional monomer.     

Injectable, mixed natural-synthetic polymer hydrogels with modular properties

A series of synthetic oligomers (based on the thermosensitive polymer poly(N-isopropylacrylamide) and carbohydrate polymers (including hyaluronic acid, carboxymethyl cellulose, dextran, and methylcellulose) were functionalized with hydrazide or aldehyde functional groups and mixed using a double-barreled syringe to create in situ gelling, hydrazone-cross-linked hydrogels. By mixing different numbers and ratios of different reactive oligomer or polymer precursors, covalently cross-linked hydrogel networks comprised of different polymeric components are produced by simple mixing of reactive components, without the need for any intermediate chemistries (e.g., grafting). In this way, hydrogels with defined swelling, degradation, phase transition, drug binding, and mechanical properties can be produced with properties intermediate to those of the mixture of reactive precursor polymers selected. When this modular mixing approach is used, one property can (in many cases) be selectively modified while keeping other properties constant, providing a highly adaptable method of engineering injectable, rapidly gelling hydrogels for potential in vivo applications.     

Overcoming limiting side reactions associated with an NHS-activated precursor of polymethacrylamide-based polymers

Combinatorial polymer libraries have recently gained popularity for the development of novel materials for a variety of biomedical applications including non-viral gene delivery systems and biodegradable polymers for tissue engineering. To streamline the nontrivial task of library synthesis, activated ester homopolymers have been used to serve as a backbone to which primary amine-containing functional groups (NH2-FGs) can be covalently bound at varying ratios. Polymethacryloxysuccinimide (poly(MAOS)) is one such homopolymer that was previously reported to be an attractive precursor for polymeric drug and gene delivery systems. The reported functionalization protocols entailed conjugating the precursor with 2 equiv of the NH2-FG at a reaction concentration of 25 mg poly(MAOS)/150 microL DMSO for either 5 h at 50 degrees C or 16 h at 25 degrees C. More recently, both protocols were revealed to be associated with ring-opening and glutarimide-forming side reactions that compromise the utility of the homopolymer. Using 1-dimensional and 2-dimensional NMR spectroscopy techniques, we have characterized the side product distributions that result from conjugations performed at 50 degrees C/5 h and 25 degrees C/16 h. Moreover, by systematically altering the equivalents of the NH2-FGs, polymer concentration, reaction time, and reaction temperature, we have established a protocol that overcomes these side reactions. Using a final reaction protocol of 5 equiv of the NH2-FG at a reaction concentration of 25 mg poly(MAOS)/600 microL DMSO for 24 h at 75 degrees C, we have obtained functionalized polymers with minimal side products. This protocol is applicable for polymers ranging from 5000 to 50,000 g/mol, compatible with a variety of functional groups, and amenable to conjugating combinations of functional groups.     

Polymerization of polyfunctional macromolecules: synthesis of a new class of high molecular weight poly(amino acid)s by oxidative coupling of phenol-containing precursor polymers

Oxidative coupling of phenol-containing precursor poly(amino acid)s, poly(alpha-glutamine), poly(alpha/beta-asparagine), and poly(gamma-glutamine) derivatives, has been examined to produce a new class of soluble poly(amino acid)s. Under appropriate reaction conditions, the Fe-salen and HRP catalysts efficiently induced the oxidative coupling without formation of insoluble gels, yielding the soluble polymers of high molecular weight. The oxidative coupling behaviors were greatly influenced by the structure and phenol content of the precursor polymer. The selection of the substrate concentration and catalyst amount was crucial for the production of soluble polymers of high molecular weight.     

Synthesis of versatile thiol-reactive polymer scaffolds via RAFT polymerization

Well-defined polymer scaffolds convertible to (multi)functional polymer structures via selective and efficient modifications potentially provide an easy, versatile, and useful approach for a wide variety of applications. Considering this, a homopolymer scaffold, poly(pyridyldisulfide ethylmethacrylate) (poly(PDSM)), having pendant groups selectively reactive with thiols, was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization. Soluble polymers with controlled molecular weights and narrow PDIs were generated efficiently. The versatility of the scaffold to generate random co- and ter-polymers combining multiple functionalities with controlled-composition was shown by separate and simultaneous conjugation of different mercapto-compounds, including a tripeptide in one-step. Conversion of water-insoluble scaffold to peptide-containing water-soluble copolymers was observed to yield nanometer-size particles with narrow polydispersity. The overall results suggest that the well-defined PDSM homopolymer scaffold generated via RAFT polymerization can be a versatile building block for generation of new structures having potential for drug delivery applications via a straightforward synthetic approach.     

Squaric acid mediated synthesis and biological activity of a library of linear and hyperbranched poly(glycerol)-protein conjugates

Polymer-protein conjugates generated from side chain functional synthetic polymers are attractive because they can be easily further modified with, for example, labeling groups or targeting ligands. The residue specific modification of proteins with side chain functional synthetic polymers using the traditional coupling strategies may be compromised due to the nonorthogonality of the side-chain and chain-end functional groups of the synthetic polymer, which may lead to side reactions. This study explores the feasibility of the squaric acid diethyl ester mediated coupling as an amine selective, hydroxyl tolerant, and hydrolysis insensitive route for the preparation of side-chain functional, hydroxyl-containing, polymer-protein conjugates. The hydroxyl side chain functional polymers selected for this study are a library of amine end-functional, linear, midfunctional, hyperbranched, and linear-block-hyperbranched polyglycerol (PG) copolymers. These synthetic polymers have been used to prepare a diverse library of BSA and lysozyme polymer conjugates. In addition to exploring the scope and limitations of the squaric acid diethylester-mediated coupling strategy, the use of the library of polyglycerol copolymers also allows to systematically study the influence of molecular weight and architecture of the synthetic polymer on the biological activity of the protein. Comparison of the activity of PG-lysozyme conjugates generated from relatively low molecular weight PG copolymers did not reveal any obvious structure-activity relationships. Evaluation of the activity of conjugates composed of PG copolymers with molecular weights of 10000 or 20000 g/mol, however, indicated significantly higher activities of conjugates prepared from midfunctional synthetic polymers as compared to linear polymers of similar molecular weight.     

A holographic sensor based on a rationally designed synthetic polymer

A new silver halide-containing holographic recording material has been designed and developed specifically for holographic chemical sensors. The hologram enables very small volume changes to be measured in a polymer layer throughout which the hologram is located. The holographic film is based on a fine-grain silver bromide emulsion suspended in a poly(vinyl alcohol) matrix crosslinked with Cr(III) ions. Cross-linking gives the material sufficient spatial integrity to allow a holographic image to be recorded, while maintaining adequate porosity and elasticity of the polymer matrix for sensing applications. The new material has been characterized with respect to its response to pH and compared with a traditional gelatin holographic film. The response to some ions and small molecules typically found in analytical samples has also been measured. Functional groups introduced covalently into the poly(vinyl alcohol) matrix transform the base matrix into a pH-responsive polymer with predictable swelling properties and which can be further derivatized to incorporate specific ligands. A rationally designed holographic sensor for trypsin has been developed from chemically synthesized artificial polymers. A trypsin substrate, the poly(amino acid) poly(L-lysine), was incorporated into poly(vinyl alcohol) holograms to create a 'designed' holographic material which was degraded in a concentration-dependent manner by trypsin. Extensions of this approach to other hydrolytic enzymes are briefly discussed.     

Designer protein-based performance materials

Repeat sequence protein polymer (RSPP) technology provides a platform to design and make protein-based performance polymers and represents the best nature has to offer. We report here that the RSPP platform is a novel approach to produce functional protein polymers that have both biomechanical and biofunctional blocks built into one molecule by design, using peptide motifs. We have shown that protein-based designer biopolymers can be made using recombinant DNA technology and fermentation and offer the ability to screen for desired properties utilizing the tremendous potential diversity of amino acid combinations. The technology also allows for large-scale manufacturing with a favorable fermentative cost-structure to deliver commercially viable performance polymers. Using three diverse examples with antimicrobial, textile targeting, and UV-protective agent, we have introduced functional attributes into structural protein polymers and shown, for example, that the functionalized RSPPs have possible applications in biodefense, industrial biotechnology, and personal care areas. This new class of biobased materials will simulate natural biomaterials that can be modified for desired function and have many advantages over conventional petroleum-based polymers.     

Gene delivery properties of end-modified poly(beta-amino ester)s

Here, we present the synthesis of a library of end-modified poly(beta-amino ester)s and assess their utility as gene delivery vehicles. Polymers were synthesized using a rapid, two-step approach that involves initial preparation of an acrylate-terminated polymer followed by a postpolymerization amine-capping step to generate end-functionalized polymers. Using a highly efficient poly(beta-amino ester), C32, we show that the terminal amine can greatly affect and improve polymer properties relevant to gene delivery. Specifically, the in vitro transfection levels can be increased by 30% and the optimal polymer:DNA ratio lowered 5-fold by conjugation of the appropriate end group. The most effective modifications were made by grafting primary diamine molecules to the chain termini. The added charge and hydrophobicity of some derivatives enhanced DNA binding and resulted in the formation of polymer-DNA complexes less than 100 nm in diameter. In addition, cellular uptake was improved 5-fold over unmodified C32. The end-modified poly(beta-amino ester)s presented here are some of the most effective gene-delivery polycations, superior to polyethylenimine and previously reported poly(beta-amino ester)s. These results show that the end-modification of poly(beta-amino ester)s is a general strategy to alter functionality and improve the delivery performance of these materials.     

Synthesis of poly(ADP-ribose)-agarose beads: an affinity resin for studying (ADP-ribose)n-protein interactions.

Polymers of ADP-ribose bind chromatosomal histones in solution and may play a role in chromatin accessibility in vivo. We have enzymatically synthesized a poly(ADP-ribose) affinity resin to further characterize binding of nuclear proteins to ADP-ribose polymers. NAD+- and (ADP-ribose)-derivatized agarose beads were recognized as polymer acceptors by the nuclear enzyme poly(ADP-ribose) polymerase. This polymerase elongated the existing ligands by successive addition of exogenously available ADP-ribose residues to form polymers covalently linked to the agarose beads. Poly(ADP-ribose) formation on the beads was dependent on incubation time and the mode of ligand attachment to the agarose. The resulting poly(ADP-ribose)-derivatized agarose beads possessed polymers which closely resembled those modifying the ADP-ribose polymerase by the automodification reaction. Fractionation of rat liver nuclear lysate over the poly(ADP-ribose) resin revealed a strong affinity of H1 for ADP-ribose polymers, thereby supporting a role for poly(ADP-ribose) in chromatin functions. Poly(ADP-ribose)-agarose beads are extremely stable and will be useful not only for affinity studies, but also for mechanistic studies involving polymer elongation and catabolism.     

Folic acid conjugated amino acid-based star polymers for active targeting of cancer cells

Amino acid-based core cross-linked star (CCS) polymers (poly(L-lysine)(arm)poly(L-cystine)(core)) with peripheral allyl functionalities were synthesized by sequential ring-opening polymerization (ROP) of amino acid N-carboxyanhydrides (NCAs) via the arm-first approach, using N-(trimethylsilyl)allylamine as the initiator. Subsequent functionalization with a poly(ethylene glycol) (PEG)-folic acid conjugate via thiol-ene click chemistry afforded poly(PEG-b-L-lysine)(arm)poly(L-cystine)(core) stars with outer PEG coronas decorated with folic acid targeting moieties. Similarly, a control was prepared without folic acid, using just PEG. A fluorophore was used to track both star polymers incubated with breast cancer cells (MDA-MB-231) in vitro. Confocal microscopy and flow cytometry revealed that the stars could be internalized into the cells, and higher cell internalization was observed when folic acid moieties were present. Cytotoxicity studies indicate that both stars are nontoxic to MDA-MB-231 cells at concentrations of up to 50 μg/mL. These results make this amino acid-based star polymer an attractive candidate in targeted drug delivery applications including chemotherapy.     

Capillary electrophoresis analysis of poly(ethylene glycol) and ligand-modified polylysine gene delivery vectors

Cationic polymers including polylysine (PLL) and polyethylenimine are being widely tested as gene delivery vectors in various gene therapy applications. In many cases, the polymers were further modified by hydrophilic polymer grafting or ligand conjugation, which had been shown to greatly affect the vector stability, delivery efficiency and specificity. The characterization of modified polycation is particularly critical for quality control and vector development. Here several different separation modes using capillary electrophoresis for the analytical characterization of the modified polymers are described. PLL molecules were grafted with poly(ethylene glycol) (PEG) chain or conjugated with epidermal growth factor and analyzed under various analytical conditions. Poly(N,N'-dimethylacrylamide)-coated capillary was used to analyze the modified PLL to reduce the interaction between the samples and the capillary wall. PLLs containing different numbers of conjugated ligands were well separated with the coating method but, for PLL-g-PEG, the separation was poor under the same conditions. A method using low buffer pH and hydroxypropylmethyl cellulose additive was developed. These methods are useful to characterize various polycations and important for the quality control and application of potential gene delivery vectors.     

Synthesis of multifunctional polyvinylsaccharide containing controllable amounts of biospecific ligands

In the present study, the attempt to synthesize a multibiofunctional polymeric vector to be used for construction of composite scaffolds for bone tissue engineering has been undertaken. The polymers based on 2-deoxy-2-methacrylamido- d-glucose were functionalized by a growth factor (BMP-2), GRGDSP peptide, and poly( l-lysine) using aldehyde chemistry. The covalent modification process was quantitatively studied, and a polymer conjugate containing all these ligands was formed. In addition, the impacts of coupled ligands toward the adsorption of polymers on the commercial mineral macroporous matrix Sponceram used in cell culture applications were studied.     

The alpha-glycosidic bonds of poly(ADP-ribose) are acid-labile.

The poly(ADP-ribosyl)ation system of higher eukaryotes produces multiple ADP-ribose polymers of distinct sizes which exhibit different binding affinities for histones. Although precipitation with trichloroacetic acid (TCA) is the standard procedure for isolation of poly(ADP-ribose) from biological material, we show here that poly(ADP-ribose) is not stable under acidic conditions. Storage of poly(ADP-ribose) as TCA pellets results in acid hydrolysis of polymers, the extent of which is dependent on storage time and temperature. The alpha-glycosidic, inter-residue bonds are the preferred sites of attack, thus reducing polymer sizes by integral numbers of ADP-ribose to yield artefactually more and smaller polymers than originally present. Therefore, poly(ADP-ribosyl)ation studies involving TCA precipitation, histone extraction with acids, or acidic incubations of ADP-ribose polymers must account for the impact of acids on resulting polymer populations.     

Glycine derived polymerizable cosurfactant in the synthesis of functionalized poly(butyl acrylate) nanolatexes

The approach of employing N-glycinylmaleamic acid (NGMA) as an efficient cosurfactant to provide microemulsion polymerization of butyl acrylate using a weight ratio of sodium dodecyl sulfate (SDS)/butyl acrylate (BA) at 相似文献   

Facile synthesis of acid-labile polymers with pendent ortho esters

This work presents a facile approach for preparation of acid-labile and biocompatible polymers with pendent cyclic ortho esters, which is based on the efficient and mild reactions between cyclic ketene acetal (CKA) and hydroxyl groups. Three CKAs, 2-ethylidene-1,3-dioxane (EDO), 2-ethylidene-1,3-dioxolane (EDL), and 2-ethylidene-4- methyl-1,3-dioxolane (EMD) were prepared from the corresponding cyclic vinyl acetals by catalytic isomerization of the double bond. The reaction of CKAs with different alcohols and diols was examined using trace of p-toluenesulfonic acid as a catalyst. For the monohydroxyl alcohols, cyclic ortho esters were formed by simple addition of the hydroxyl group toward CKAs with ethanol showing a much greater reactivity than iso-propanol. When 1,2- or 1,3-diols were used to react with the CKAs, we observed the isomerized cyclic ortho esters besides the simple addition products. Biocompatible polyols, that is, poly(2-hydroxyethyl acrylate) (PHEA) and poly(vinyl alcohol) (PVA) were then modified with CKAs, and the degree of substitution of the pendent ortho esters can be easily tuned by changing feed ratio. Both the small molecule ortho esters and the CKA-modified polymers demonstrate the pH-dependent hydrolysis profiles, which depend also on the chemical structure of the ortho esters as well as the polymer hydrophobicity.     

A novel reductive graphene oxide‐based magnetic molecularly imprinted poly(ethylene‐co‐vinyl alcohol) polymers for the enrichment and determination of polychlorinated biphenyls in fish samples

The novel reductive graphene oxide‐based magnetic molecularly imprinted poly(ethylene‐co‐vinyl alcohol) polymers (rGO@m‐MIPs) were successfully synthesized as adsorbents for six kinds of polychlorinated biphenyls (PCBs) in fish samples. rGO@m‐MIPs was prepared by surface molecular imprinting technique. Besides, Fe₃O₄ nanoparticles (NPs) were employed as magnetic supporters, and rGO@Fe₃O₄ was in situ synthesis. Different from functional monomer and cross‐linker in traditional molecularly imprinted polymer, here, 3,4‐dichlorobenzidine was employed as dummy molecular and poly(ethylene‐co‐vinyl alcohol) was adopted as the imprinted polymers. After morphology and inner structure of the magnetic adsorbent were characterized, the adsorbent was employed for disperse solid phase extraction toward PCBs and exhibited great selectivity and high adsorption efficiency. This material was verified by determination of PCBs in fish samples combined with gas chromatography‐mass spectrometry (GC‐MS) method. According to the detection, the low detection limits (LODs) of PCBs were 0.0035–0.0070 µg l⁻¹ and spiked recoveries ranged between 79.90 and 94.23%. The prepared adsorbent can be renewable for at least 16 times and expected to be a new material for the enrichment and determination of PCBs from contaminated fish samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis and biologically relevant properties of polyphosphazene polyacids

Polyphosphazene polyacids show potential as immunostimulating compounds and materials for microencapsulation. Their synthesis requires multistep chemical transition from a hydrolytically unstable macromolecular precursor, poly(dichlorophosphazene), to a water-soluble polyelectrolyte. Insufficient synthetic control in these reactions can lead to molecular weight variations and formation of macromolecules with "structural defects" resulting in significant variations in polymer performance. Simple and reproducible "one pot-one solvent" method is reported for the preparation of polyphosphazene polyacids-poly[di(carboxylatophenoxy)phosphazene] and its copolymers. Molecular weight characteristics and polymer compositions were studied as a function of reaction parameters. Macromolecular byproducts, incompletely substituted polymers containing hydroxyl groups and partially deprotected polymers containing propyl ester functionalities, were synthesized and characterized. It was demonstrated, that the presence of such groups can affect polymer characteristics, such as hydrolytic degradation profiles, immunostimulating activity, and microsphere forming properties. In vivo studies showed that the immunostimulating activity of polyphosphazene polyacids correlates with the content of acid functionalities in the polymer.     

Procedure for Fabricating Biofunctional Nanofibers

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, α,ω-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of α-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The α,ω-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-∈-amino caproic acid, by DCC coupling, resulting in the formation of α,ω-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, ¹H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.     

Tumor-targeted gene delivery using molecularly engineered hybrid polymers functionalized with a tumor-homing peptide

Before gene therapy can be used in clinical settings, safe and efficient DNA delivery systems must be developed to overcome a range of extra- and intracellular transport barriers. As a step toward the development of a modular, multifunctional gene delivery system to overcome these diverse barriers, we have developed a family of linear-dendritic "hybrid" polymers which contain functionalities for tissue targeting, minimization of nonspecific interactions, endosomal buffering, and DNA binding. Here, we demonstrate the rapid three-step, room-temperature, aqueous synthesis of hybrid polymers, as well as the functionalization of these polymers with a peptide targeting ligand that specifically binds to glucose-regulated protein-78 kDa (GRP-78), a clinically relevant tumor antigen identified in human cancer patients. These polymer systems can condense plasmid DNA into small nanoparticle structures (<210 nm) and transfect cells expressing GRP-78 with efficiencies that exceed that of branched polyethylenimine (bPEI), one of the best commercially available polymers for in vitro transfections. The synthetic approach described here may be useful for the rapid synthesis and optimization of polymer gene delivery systems bearing a range of diverse functional domains, and the specific GRP-78-targeted systems developed in this study may potentially have clinical applications in cancer gene therapy.