Synthesis and characterization of biodegradable peptide-based polymers prepared by microwave-assisted click chemistry

In this study, the microwave-assisted copper(I)-catalyzed 1,3-dipolar cycloaddition reaction was used to synthesize peptide triazole-based polymers from two novel peptide-based monomers: azido-phenylalanyl-alanyl-lysyl-propargyl amide (1) and azido-phenylalanyl-alanyl-glycolyl-lysyl-propargyl amide (2). The selected monomers have sites for enzymatic degradation as well as for chemical hydrolysis to render the resulting polymer biodegradable. Depending on the monomer concentration in DMF, the molecular mass of the polymers could be tailored between 4.5 and 13.9 kDa (corresponding with 33-100 amino acid residues per polymer chain). As anticipated, both polymers can be enzymatically degraded by trypsin and chymotrypsin, whereas the ester bond in the polymer of 2 undergoes chemical hydrolysis under physiological conditions, as was shown by a ninhydrin-based colorimetric assay and MALDI-TOF analysis. In conclusion, the microwave-assisted copper(I)-catalyzed 1,3-dipolar cycloaddition reaction is an effective tool for synthesizing biodegradable peptide polymers, and it opens up new approaches toward the synthesis of (novel) designed biomedical materials.     

Development and application of thermo-sensitive immunomicrospheres for antibody purification

The latex particles composed of poly(styrene/N-isopropylacrylamide/glycidyl methacrylate) [P(St/NIPAM/GMA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] were prepared by emulsifier-free emulsion polymerization. These latex particles with submicrometer size showed the thermosensitivity originated from the thermo-sensitive nature of NIPAM. That is, the minimum NaCI concentration for flocculation of these latex particles [critical flocculation concentration (CFC)] decreased significantly with increasing temperature and reached constant values at above the critical temperature [critical flocculation temperature (CFT)]. At a certain NaCl concentration, the thermo-sensitive latex particles were flocculated by raising temperature, and conversely, the flocculated thermo-sensitive latex particles were completely dispersed by lowering temperature. Bovine serum albumin (BSA) was covalently immobilized onto the P(St/NIPAM/GMA) and P(St/NIPAM/MMA) latex particles with high efficiency. The BSA-immobilized P(St/NIPAM/GMA) and P(St/NIPAM/MAA) latex particles (immunomicrospheres) showed the similar dependencies of CFC on temperature to the bare latex particles. These thermo-sensitive immunomicrospheres were successfully used for the immunoaffinity purification of anti-BSA antibodies from antiserum. (c) 1994 John Wiley & Sons, Inc.     

Engineering temperature-sensitive poly(N-isopropylacrylamide) polymers as carriers of therapeutic proteins

This study was carried out to engineer N-isopropylacrylamide (NiPAM) polymers that contain protein-reactive N-acryloxysuccinimide (NASI) and hydrophobic alkylmethacrylates (AMAs). These thermoreversible, protein-conjugating polymers hold potential for retention of therapeutic proteins at an application site where tissue regeneration is desired. The lower critical solution temperatures (LCST) of the polymers were effectively controlled by the AMA mole content. The AMAs with longer side-chains were more effective in lowering the LCST. Polymers without NASI exhibited a stable LCST in phosphate buffer and in serum over a 10-day study period. The LCST of polymers containing NASI was found to increase over time in phosphate buffer, but not in serum-containing medium. The LCST increase in phosphate buffer was proportional to the AMA content. The feasibility of localizing a therapeutic protein, recombinant human bone morphogenetic protein-2 (rhBMP-2), to a site of application was explored in a rat intramuscular injection model. The results indicated that polymers capable of conjugating to rhBMP-2 were most effective in localizing the protein irrespective of the LCST (13-25 degrees C). For polymers with no NASI groups, a lower LCST resulted in a better rhBMP-2 localization. We conclude that thermosensitive polymers can be engineered for delivery of therapeutic proteins to improve their therapeutic efficacy.     

N-isopropylacrylamide as a functional monomer for noncovalent molecular imprinting

Although N-isopropylacrylamide (NIPAM) has previously been used in molecular imprinting, it has mostly been considered as an 'inert' monomer, or included for its temperature-responsive nature, rather than as a functional monomer responsible for the interactions with the template at the recognition site. A comparative study of NIPAM and other traditional, functional monomers for the imprinting of a hydrogen bond donor template, bisphenol A (BPA), is reported. Nuclear magnetic resonance titration data suggest that NIPAM forms a stronger complex with BPA than either acrylamide or methacrylic acid but a weaker complex than vinylpyridine. Molecular imprinted polymers (MIPs) were prepared using each functional monomer and compared as stationary phases for the separation of BPA from structural analogues. The NIPAM-containing MIP bound BPA with better selectivity than those prepared using acrylamide or methacrylic acid. Using NIPAM also reduces the nonspecific binding, which is found with MIPs using vinylpyridine as functional monomer.     

Highly branched poly(N-isopropylacrylamide) for use in protein purification

Poly(N-isopropylacrylamide)s with imidazole endgroups were used to separate a histidine-tagged protein fragment directly from a crude cell lysate. The polymers display a lower critical solution temperature that can be tuned to occur at a range of subambient temperatures. UV-visible spectra indicated differences in the binding in aqueous media of Cu(II) and Ni(II) to the imidazole endgroups. These changes in the UV-visible spectra were reflected in the solution/aggregation behavior of the polymers as studied by dynamic light scattering. The addition of Cu(II) disaggregated the polymers, and the polymer coil swelled. On the other hand, when Ni(II) was added the polymers remained aggregated in aqueous media. The polymers were used to purify residues 230-534 of the histidine-tagged breast cancer susceptibility protein his6-BRCA1. Cu(II) was found to be better suited to the formation of useful polymer-metal ion-protein complexes that display cloud points, since Ni(II)/polymer mixtures generated very little purified protein. The polymers were synthesized using a previously reported variation of the reversible addition-fragmentation chain termination (RAFT) methodology, using the chain transfer agent 3H-imidazole-4-carbodithioic acid 4-vinyl benzyl ester with N-isopropylacrylamide (NIPAM).     

Analysis of the interactions between Eudragit® L100 and porcine pancreatic trypsin by calorimetric techniques

Flexible-chain polymers with charge (polyelectrolytes) can interact with globular proteins with a net charge opposite to the charge of the polymers forming insoluble complexes polymer-protein. In this work, the interaction between the basic protein trypsin and the anionic polyelectrolyte Eudragit^® L100 was studied by using isothermal calorimetric titrations and differential scanning calorimetry. Turbidimetric assays allowed determining that protein-polymer complex was insoluble at pH below 5 and the trypsin and Eudragit^® L100 concentrations required forming the insoluble complex. DSC measurements showed that the T_m and denaturalization heat of trypsin increased in the polymer presence and the complex unfolded according to a two-state model. ΔH° and ΔS° binding parameters obtained by ITC were positives agree with hydrophobic interaction between trypsin and polymer. However, ionic strength of 1.0 M modified the insoluble complex formation. We propose a mechanism of interaction between Eudragit^® L100 and trypsin molecules that involves both hydrophobic and electrostatic interactions. Kinetic studies of complex formation showed that the interaction requires less than 1 min achieving the maximum quantity of complex. Finally, a high percentage of active trypsin was precipitated (approximately 76% of the total mass of protein). These findings could be useful in different protocols such as a protein isolation strategy, immobilization or purification of a target protein.     

Precipitation with poly acrylic acid as a trypsin bioseparation strategy

Precipitation of enzymes with reversible soluble–insoluble polymers is a simple approach which can be easily scaled up. This work reports investigations aiming at verifying the existence of specific interactions and complex formation between porcine trypsin and poly acrylic acids using spectroscopy techniques. The trypsin–polymer complex was insoluble at pH lower than 5, with a stoichiometric ratio polymer mol per protein mol of 1:148. It took only a minute for the insoluble complex to form and it was redissolved modifying the pH of the medium. The enzymatic activity of trypsin was maintained even in the presence of the polymer and after precipitation poly acrylic acid presence protect the enzyme from itself degradation. The conditions of complex formation were studied using pure proteins that could be applied on porcine pancreas homogenates as an isolation strategy of trypsin.     

Characterization of structural component of cell walls of alkalophilic strain of Bacillus sp. C-125. Preparation of poly(gamma-L-glutamate) from cell wall component.

The cell wall of an alkalophilic strain of Bacillus sp. C-125 is composed of A1 gamma-peptidoglycan, a teichuronic acid and an unknown acidic polymer composed of glutamic acid and glucuronic acid, of which the molar ratio is approx. 4-5:1. Poly(gamma-L-glutamate) was prepared from the acidic polymer by removal of almost all of the glucuronic residues with trifluoromethanesulphonic acid treatment and purified chromatographically. The Mr of the polyglutamate preparation was estimated to be 14,000 by gel chromatography, or 43,000 on the basis of the content of N-terminal acid residues. The acidic polymer found in the cell wall of the organism was concluded to be a polyglutamate substituted with (oligo)glucuronic acid residues or a complex composed of two kinds of polymers (polyglutamate and polyglucuronate).     

A continuous affinity ultrafiltration process for trypsin purification

A continuous process has been devised and tested for purification of a crude trypsin preparation from pig pancreas. The development was based on the principles of affinity chromatography and Ultrafiltration. Trypsin was selectively attracted by a water-soluble high molecular weight (>100,000) polymer, bearing a potent and specific trypsin inhibitor, m-aminobenzamidine. The trypsin-macroligand complex was then retained by using an appropriate Ultrafiltration membrane, while impurities could pass through. The bound trypsin was eluted by either arginine or benzamidine. The process also featured provision for recirculation of the eluant as well as the macroligand. It was demonstrated that this purification process could purify trypsin from the crude preparation with a yield of 77%, contaminated with only 3% of impurities. For the first time, a serious attempt has been made toward continuous purification of enzymes by the affinity Ultrafiltration technique. Besides a substantial increase in productivity, the affinity polymer could be easily reconditioned and expected to possess a long operative life. Such characteristics undoubtedly will play an important role in reducing the cost of trypsin purification.     

Affinity chromatography of trypsin and related enzymes. I. Preparation and characteristics of an affinity adsorbent containing tryptic peptides from protamine as ligands.

An absorbent for the affinity chromatography of trypsin [EC 3.4.21.4] (AP Sepharose) was prepared. The ligand was a mixture of oligopeptides (mainly di- and tripeptides) containing L-arginine as carboxyl termini, and was obtained from a tryptic digest of protamine. Trypsin was absorbed at relatively low pH (7-4), but was not absorbed at the optimum pH of catalysis (8.2). This was clearly explained on the basis of the pH dependence of the interaction of trypsin with its products. Inactivated trypsin, trypsinogen, and chymotrypsin were not absorbed. The absorption of active trypsin was interferred with by either benzamidine or urea. From these observations, it is evident that AP Sepharose is an affinity adsorbent. AP Sepharose was useful for purification of commercial bovine trypsin. A preliminary application for the purification of Streptomyces griseus trypsin was also successful.     

Enhancing ligand–protein binding in affinity thermoprecipitation: Elucidation of spacer effects

Copolymers of N‐isopropylacrylamide and N‐acryloyl amino acid spacers of varying chain length were synthesized. p‐Aminobenzamidine (PABA) was chemically linked to the pendant carboxyl groups of these polymers to obtain thermoprecipitating affinity polymers. The inhibition constant (K_i) of these polymers for trypsin decreased, i.e., the efficiency of PABA–trypsin binding increased with increase in the spacer chain length. The polymer to which PABA was linked through a spacer of five methylene groups exhibited eleven times lower K_i than that of the polymer containing PABA without a spacer. Investigations on model inhibitors N‐acyl‐p‐aminobenzamidines showed that this enhancement in trypsin binding by the polymers was due to the spacer as well as to microenvironmental effects. Recovery and specific activity of the trypsin recovered increased with the spacer chain length. Separation of trypsin from a mixture of trypsin and chymotrypsin was also enhanced with the spacer chain length. The inhibition constants of these affinity polymers were not adversely affected by the crowding effect. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 418–425, 1999.     

Preparation and tumor cell uptake of poly(N-isopropylacrylamide) folate conjugates

Folate conjugates (PNIPAM-NH-FA) of a copolymer of N-isopropylacrylamide (NIPAM) and amino-N'-ethylenedioxy-bis(ethylacrylamide) were prepared by an efficient synthesis leading to random grafting, via a short dioxyethylene spacer, of approximately 7 folic acid residues per macromolecule. The chemical composition of the copolymer was characterized by (1)H NMR and UV/vis spectroscopy. A fluorophore-labeled folate PNIPAM conjugate was tested by in vitro assays performed with cultured KB-31 cells overexpressing the folate receptor. The cellular uptake of the copolymer was found to be temperature dependent and was competitively decreased by free folic acid, indicating that the polymer uptake is mediated specifically by the folate receptor. Hydrophobically modified folate conjugates of NIPAM, amino-N'-ethylenedioxy-bis(ethylacrylamide) copolymers, bearing a small number of n-octadecyl groups were prepared following a modified synthetic procedure for use in future studies of FA-targeted liposomes.     

Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase.

Recent results revealed that inorganic polyphosphates (polyP), being energy-rich linear polymers of orthophosphate residues known from bacteria and yeast, also exist in higher eukaryotes. However, the enzymatic basis of their metabolism especially in mammalian cells is still uncertain. Here we demonstrate for the first time that alkaline phosphatase from calf intestine (CIAP) is able to cleave polyP molecules up to a chain length of about 800. The enzyme acts as an exopolyphosphatase degrading polyP in a processive manner. The pH optimum is in the alkaline range. Divalent cations are not required for catalytic activity but inhibit the degradation of polyP. The rate of hydrolysis of short-chain polyP by CIAP is comparable to that of the standard alkaline phosphatase (AP) substrate p-nitrophenyl phosphate. The specific activity of the enzyme decreases with increasing chain length of the polymer both in the alkaline and in the neutral pH range. The K(m) of the enzyme also decreases with increasing chain length. The mammalian tissue non-specific isoform of AP was not able to hydrolyze polyP under the conditions applied while the placental-type AP and the bacterial (Escherichia coli) AP displayed polyP-degrading activity.     

Affinity precipitation of alpha-amylase inhibitor from wheat meal by metal chelate affinity binding using cu(II)-loaded copolymers of 1-vinylimidazole with N-isopropylacrylamide

A method for purifying alpha-amylase inhibitor from wheat meal based on immobilized metal affinity with a thermosensitive copolymer is developed. The studies represent the thermoprecipitation properties of the copolymers of N-isopropylacrylamide (NIPAM) with iminodiacetic acid (IDA) and 1-vinylimidazole (VI), respectively. The polymer which is obtained by the copolymerization of 1-vinylimidazole and N-isopropylacrylamide, charged with Cu(II), exhibited specific interaction of the metal ions to the protein inhibitor. The precipitation was induced by salt and the recovery of the amylase inhibitor was achieved by dissolving the inhibitor-polymer complex in imidazole buffer and subsequent precipitation of the polymer. A single family of the alpha-amylase inhibitor was recovered from the polymer with 89% yield and about fourfold purification. The SDS-PAGE pattern showed significant purification of the inhibitor. The binding of the inhibitor to the Cu(II)-polymer conjugate depends upon the Cu(II) concentration in the copolymer and also upon the concentration of the protein. The recovered polymer could be reused with reasonable efficiency. Copyright 1998 John Wiley & Sons, Inc.     

Design and expression of polymeric immunoglobulin fusion proteins: a strategy for targeting low-affinity Fcgamma receptors

We have developed a family of cloning vectors that direct expression of fusion proteins that mimic aggregated immunoglobulin (IgG) (AIG) and immune complex function with respect to their interactions with FcgammaR and that allow for the inclusion and targeting of a second protein domain to cells expressing FcgammaR. This was accomplished by expressing multiple linear copies of the hinge and CH2 domains (HCH2) of human IgG(1) fused to the framework region of human IgG(1). Convenient restriction sites allow for the facile introduction of additional amino-terminal domains. The resulting molecule is tripartite. The carboxyl-IgG(1) framework domain provides stability and permits dimerization, and the intervening polymer provides increased effector function and targeting to FcgammaR while the amino-terminal domain can deliver an additional signal to cells expressing FcgammaR. To demonstrate the utility of the vectors, the extracellular domain of human CD8alpha was expressed as a HCH2 polymer fusion protein. The fusion proteins were secreted in useful amounts from polyclonal cell lines established in Sf9 cells following transfection and selection with G418. The biological activity of the recombinant CD8alpha-HCH2 polymers was determined and compared to those of AIG and an anti-CD16 monoclonal antibody using an in vitro assay. The activity of the fusion proteins positively correlates to the number of HCH2 units. The largest polymer tested was severalfold more potent than AIG at similar concentrations. The HCH2 polymers described here represent a new strategy in the design of recombinant proteins for the therapeutic targeting of FcgammaR in autoimmune disorders.     

Affinity precipitation of proteins: design criteria for an efficient polymer

Affinity precipitation is fast emerging as a successful technique for the purification of proteins which can be introduced at an early stage of downstream processing. The technique applies the use of reversibly soluble-insoluble polymers which have either natural or synthetic origin. Apart from the successful use of some natural polymers, such as chitosan and alginate, the vast application of the technique depends upon the design of efficient synthetic polymers. In this laboratory, N-isopropylacrylamide (NIPAM) copolymers have been developed for metal chelate affinity precipitation of proteins. The copolymers of 1-vinylimidazole (VI) and iminodiacetic acid (IDA) with NIPAM were synthesized. The copolymers were thoroughly characterized with a view to designing an efficient soluble-insoluble polymer for metal chelate affinity precipitation of proteins.     

New biodegradable hydrogels based on a photocrosslinkable modified polyaspartamide: synthesis and characterization.

alpha,beta-Poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA), a synthetic water-soluble biocompatible polymer, was derivatized with glycidyl methacrylate (GMA), in order to introduce in its structure chemical residues having double bonds and ester groups. The obtained copolymer (PHG) contained 29 mol% of GMA residues. PHG aqueous solutions at various concentrations ranging from 30 to 70 mg/ml were exposed to a source of UV rays at lambda 254 nm in the presence or in the absence of N,N'-methylenebisacrylamide (BIS); the formation of compact gel phases was observed beginning from 50 mg/ml. The obtained networks were characterized by FT-IR spectrophotometry and swelling measurements which evidenced the high affinity of PHG hydrogels towards aqueous media at different pH values. In vitro chemical or enzymatic hydrolysis studies suggested that the prepared samples undergo a partial degradation both at pH 1 and pH 10 and after incubation with enzymes such as esterase, pepsin and alpha-chymotrypsin. Finally, the effect of irradiation time on the yield and the properties of these hydrogels was investigated and the sol fractions coming from irradiated samples, properly purified, were characterized by FT-IR and 1H-NMR analyses.     

Structure of artificial cytoskeleton containing liposomes in aqueous solution studied by static and dynamic light scattering

The structure of three types of liposomes (egg yolk phosphatidylcholine (EPC) without modification and EPC vesicles containing cross-linked N-isopropylacrylamide (NIPAM) networks of low and a high concentration inside the vesicles) were analyzed by static and dynamic light scattering. Upon polymerization the network was assumed to become attached to the membrane by reactive anchoring monomers. For the sample of high poly(NIPAM) content the polymer network was assumed to fill the whole space in the vesicles. The issue of the present study was to examine hard and hollow sphere behavior of the liposomes with networks of high and low poly(NIPAM) content. The theoretical scattering curves differ markedly for uniform hard and uniform hollow spheres by the presence of specific peaks. However, polydispersity washed out the peaks and led to smoothed asymptotes with fractal dimensions of df = 2 for hollow and df = 4 for hard spheres. The experimental data could efficiently be fitted with weakly polydisperse hollow spheres. No clear conclusion could be drawn from the angular dependence alone for the liposome of high poly(NIPAM) content. The two wavelengths from the HeNe and Ar lasers proved to be too long for the studied liposomes of about 100 nm in radius. However, evidence for hollow sphere behavior was found for fractionated liposomes from the ratio rho = Rg/Rh = 1.04 +/- 0.02 (theory rho = 1.00 for hollow spheres). Finally, from the molar mass and the sphere radius, an apparent density was determined. The analysis gave the expected density for the pure EPC lecithin vesicles and a poly(NIPAM) network density of 0.244 g/mL. For the liposome of low poly(NIPAM) content the network appeared to be attached to the inner surface of the lecithin shell to form a layer of about 18 nm thickness.     

Interaction of sugars, polysaccharides and cells with boronate-containing copolymers: from solution to polymer brushes

Interaction of mono- and disaccharides, polysaccharide particles and yeast cells with boronate-containing copolymers (BCC) of N-acryloyl-m-aminophenylboronic acid (NAAPBA) with N,N-dimethylacrylamide (DMAA) or N-isopropylacrylamide (NIPAM) was studied. The binding of saccharides to BCC of NIPAM resulted in a shift of its phase transition temperature (DeltaTP), which provided a quantitative measure for the complex formation. Among the sugars typical of non-reducing ends of glycoproteins the DeltaTP decreased in the order: N-acetylneuraminic acid > xylose approximately galactose > mannose approximately fucose > N-acetylglucosamine. Strong specific adsorption of the BCC on the cross-linked agarose gel Sepharose CL-6B (15-30 mg/ml gel at pH 9.2) was registered. The copolymers adsorption was due to boronate-sugar interactions and decreased with pH. Multivalent interaction of the BCC with the agarose gel has been proven by liquid column chromatography exhibiting a weak reversible adsorption of NAAPBA and almost irreversible adsorption of DMAA-NAAPBA copolymer from 0.1 M sodium phosphate buffer, pH 7.9. The two studied BCCs could be completely desorbed from the gel by 0.1 M fructose in aqueous buffered media with pH from 7.5 to 9.2. In turn, the agarose particles and yeast cells were found to adhere to siliceous supports end-grafted with boronate-BCC of N,N-dimethylacrylamide at pH > or = 7.5, due to the actions. Quantitative detachment of adhered particles or cells could be attained by addition of 20 mM or 100 mM fructose, respectively, in the pH range from 7.5 to 9.2. Affinity adhesion of micron-size carbohydrate particles to boronate-containing polymer brushes fixed on solid supports was considered as a model system suggesting a new approach to isolation and separation of living cells.