Synthetic Glycopolymer Research Articles

Poly(2-Deoxy-2-Methacrylamido-D-Glucose)-Based Complex Conjugates of Colistin, Deferoxamine and Vitamin B12: Synthesis and Biological Evaluation.

Growing resistance to traditional antibiotics poses a global threat to public health. In this regard, modification of known antibiotics, but with limited applications due to side effects, is one of the extremely promising approaches at present. In this study, we proposed the synthesis of novel complex polymeric conjugates of the peptide antibiotic colistin (CT). A biocompatible and water-soluble synthetic glycopolymer, namely, poly(2-deoxy-2-methacrylamido-D-glucose) (PMAG), was used as a polymer carrier. In addition to monoconjugates containing CT linked to PMAG by hydrolyzable and stable bonds, a set of complex conjugates also containing the siderophore deferoxamine (DFOA) and vitamin B12 was developed. The structures of the conjugates were confirmed by 1H NMR and FTIR-spectroscopy, while the compositions of conjugates were determined by UV-Vis spectrophotometry and HPLC analysis. The buffer media with pH 7.4, corresponding to blood or ileum pH, and 5.2, corresponding to the intestinal pH after ingestion or pH in the focus of inflammation, were used to study the release of CT. The resulting conjugates were examined for cytotoxicity and antimicrobial activity. All conjugates showed less cytotoxicity than free colistin. A Caco-2 cell permeability assay was carried out for complex conjugates to simulate the drug absorption in the intestine. In contrast to free CT, which showed very low permeability through the Caco-2 monolayer, the complex polymeric conjugates of vitamin B12 and CT provided significant transport. The antimicrobial activity of the conjugates depended on the conjugate composition. It was found that conjugates containing CT linked to the polymer by a hydrolyzable bond were found to be more active than conjugates with a non-hydrolyzable bond between CT and PMAG. Conjugates containing DFOA complexed with Fe3+ were characterized by enhanced antimicrobial activity against Pseudomonas aeruginosa compared to other conjugates.

Preparation of a water-soluble polymer having pheophorbide a side chains using glycopolymer assembly

The first trial of incorporation of pheophorbide a (Pba) into a synthetic glycopolymer giving water-soluble ability was carried out, and the polymerization reaction of each monomer proceeded in DMSO to afford the desired glycopolymer having Pba side chains. Characterization of the polymer was performed by means of spectroscopic analyses and thermal studies, and the results supported the desired macromolecular architecture.

Polymyxin B Conjugates with Bio-Inspired Synthetic Polymers of Different Nature.

The emergence and growth of bacterial resistance to antibiotics poses an enormous threat to humanity in the future. In this regard, the discovery of new antibiotics and the improvement of existing ones is a priority task. In this study, we proposed the synthesis of new polymeric conjugates of polymyxin B, which is a clinically approved but limited-use peptide antibiotic. In particular, three carboxylate-bearing polymers and one synthetic glycopolymer were selected for conjugation with polymyxin B (PMX B), namely, poly(α,L-glutamic acid) (PGlu), copolymer of L-glutamic acid and L-phenylalanine (P(Glu-co-Phe)), copolymer of N-vinyl succinamic acid and N-vinylsuccinimide (P(VSAA-co-VSI)), and poly(2-deoxy-2-methacrylamido-D-glucose) (PMAG). Unlike PGlu and PMAG, P(Glu-co-Phe) and P(VSAA-co-VSI) are amphiphilic and form nanoparticles in aqueous media. A number of conjugates with different polymyxin B loading were synthesized and characterized. In addition, the complex conjugates of PGLu or PMAG with polymyxin B and deferoxamine (siderophore) were obtained. A release of PMX B from Schiff base and amide-linked polymer conjugates was studied in model buffer media with pH 7.4 and 5.8. In both cases, a more pronounced release was observed under slightly acidic conditions. The cytotoxicity of free polymers and PMX B as well as their conjugates was examined in human embryonic kidney cells (HEK 293T cell line). All conjugates demonstrated reduced cytotoxicity compared to the free antibiotic. Finally, the antimicrobial efficacy of the conjugates against Pseudomonas aeruginosa was determined and compared. The lowest values of minimum inhibitory concentrations (MIC) were observed for polymyxin B and polymyxin B/deferoxamine conjugated with PMAG. Among the polymers tested, PMAG appears to be the most promising carrier for delivery of PMX B in conjugated form due to the good preservation of the antimicrobial properties of PMX B and the ability of controlled drug release.

Functional Hybrid Glyconanocapsules by a One-Pot Nanoprecipitation Process.

Herein, we report on one-pot fabrication of oil-filled nanocapsules wrapped with both RAFT-made glycopolymers and neutral polysaccharides (dextran and pullulan). We have made use of the nanoprecipitation technique, relying on coprecipitation of both oil and polymers in conditions thoroughly established from phase diagrams' interpretation. Mixed mono- or multilayered nanocapsules were obtained through simultaneous or sequential nanoprecipitations, respectively. Incorporation of synthetic glycopolymer chains allows for precisely tailoring the dimensions of the nanocapsules (size and membrane thickness of the polymeric shell), whereas the insertion of polysaccharides enables to tune the (bio)degradability of the nanocapsules. Shell-functionalized and/or core-loaded capsules could also be achieved in a similar one-pot process, by introducing a drug and/or biotin in the organic and aqueous phase, respectively.

Bio-Inspired Amphiphilic Block-Copolymers Based on Synthetic Glycopolymer and Poly(Amino Acid) as Potential Drug Delivery Systems

In this work, a method to prepare hybrid amphiphilic block copolymers consisting of biocompatible synthetic glycopolymer with non-degradable backbone and biodegradable poly(amino acid) (PAA) was developed. The glycopolymer, poly(2-deoxy-2-methacrylamido-D-glucose) (PMAG), was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Two methods for modifying the terminal dithiobenzoate-group of PMAG was investigated to obtain the macroinitiator bearing a primary aliphatic amino group, which is required for ring-opening polymerization of N-carboxyanhydrides of hydrophobic α-amino acids. The synthesized amphiphilic block copolymers were carefully analyzed using a set of different physico-chemical methods to establish their composition and molecular weight. The developed amphiphilic copolymers tended to self-assemble in nanoparticles of different morphology that depended on the nature of the hydrophobic amino acid present in the copolymer. The hydrodynamic diameter, morphology, and cytotoxicity of polymer particles based on PMAG-b-PAA were evaluated using dynamic light scattering (DLS) and transmission electron microscopy (TEM), as well as CellTiter-Blue (CTB) assay, respectively. The redox-responsive properties of nanoparticles were evaluated in the presence of glutathione taken at different concentrations. Moreover, the encapsulation of paclitaxel into PMAG-b-PAA particles and their cytotoxicity on human lung carcinoma cells (A549) and human breast adenocarcinoma cells (MCF-7) were studied.

A glycopolymer improves vascoelasticity and mucociliary transport of abnormal cystic fibrosis mucus.

Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca2+ upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca2+-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.

Specific Inhibition of Heparanase by a Glycopolymer with Well-Defined Sulfation Pattern Prevents Breast Cancer Metastasis in Mice.

Heparanase, the heparan sulfate polysaccharide degrading endoglycosidase enzyme, has been correlated with tumor angiogenesis and metastasis and therefore has become a potential target for anticancer drug development. In this systematic study, the sulfation pattern of the pendant disaccharide moiety on synthetic glycopolymers was synthetically manipulated to achieve optimal heparanase inhibition. Upon evaluation, a glycopolymer with 12 repeating units was determined to be the most potent inhibitor of heparanase (IC50 = 0.10 ± 0.36 nM). This glycopolymer was further examined for cross-bioactivity using a solution-based competitive biolayer interferometry assay with other HS-binding proteins (growth factors, P-selectin, and platelet factor 4), which are responsible for mediating angiogenic activity, cell metastasis, and antibody-induced thrombocytopenia. The synthetic glycopolymer has low affinity for these HS-binding proteins in comparison to natural heparin. In addition, the glycopolymer possessed no proliferative properties toward human umbilical endothelial cells (HUVECs) and a potent antimetastatic effect against 4T1 mammary carcinoma cells. Thus, our study not only establishes a specific inhibitor of heparanase with high affinity but also illustrates the high effectiveness of this multivalent heparanase inhibitor in inhibiting experimental metastasis in vivo.

Controlling pre-osteoblastic cell adhesion and spreading on glycopolymer brushes of variable film thickness.

Controlling the cell behavior on biocompatible polymer surfaces is critical for the development of suitable medical implant coatings as well as in anti-adhesive applications. Synthetic glycopolymer brushes, based on sugar methacrylate monomers have been reported as robust surfaces to resist protein adsorption and cell adhesion. In this study, poly(D-gluconamidoethyl methacrylate) (PGAMA) brushes of various chain lengths were synthesized directly from initiator functionalized glass substrates using surface-initiated atom transfer radical polymerization. The glycopolymer film thicknesses were determined by ellipsometry, whereas the wettability and the morphology of the surfaces were characterized by static water contact angle measurements and atomic force microscopy, respectively. Stable, grafted films with thicknesses in the dry state between 4 and 20 nm and of low roughness (~1 nm) were obtained by varying the polymerization time. Cell experiments with MC3T3-E1 pre-osteoblasts cultured on the PGAMA brushes were performed to examine the effect of film thickness on the cell morphology, cytoskeleton organization and growth. The results revealed good cell spreading and proliferation on PGAMA layers of low film thickness, whereas cell adhesion was prevented on polymer films with thickness higher than ~10 nm, indicating their potential use in medical implants and anti-adhesive surfaces, respectively.

Poly(glycoamidoamine) Vehicles Promote pDNA Uptake through Multiple Routes and Efficient Gene Expression via Caveolae-Mediated Endocytosis

The use of synthetic polymers for the delivery of nucleic acids holds considerable promise for understanding and treating disease at the molecular level. This work aims to decipher the cellular internalization mechanisms for a series of synthetic glycopolymer DNA delivery vehicles we have termed poly(glycoamidoamine)s (PGAAs). To this end, we have performed cellular delivery experiments in the presence of pharmacological endocytosis inhibitors. Confocal microscopy analysis showed colocalization of labeled pDNA in polyplexes with immunolabeled endocytic molecules to identify the cellular internalization pathways in HeLa cells. Direct membrane penetration was also investigated through various methods, including cellular energy depletion and leakage of a cytosolic enzyme from the cell. The data suggests that the cellular internalization of PGAA polyplexes occurs through a multifaceted internalization mechanism primarily involving caveolae, yet clathrin-coated vesicles and macropinosomes were also involved to a lesser degree. The primary mechanism that leads to efficient nuclear delivery and transgene expression appears to be caveolae/raft-mediated endocytosis. The cellular internalization pathways for PGAAs were not identical to those for polyethylenimine, illustrating that differences in the chemical structure of materials directly impacts the cellular internalization mechanisms.

Epidermal Growth Factor Signaling for Matrix-Dependent Cell Proliferation and Differentiation in Primary Cultured Hepatocytes

Understanding hepatocellular signaling occurring in biomaterial systems is important for successful hepatic tissue engineering. Toward this end, we employed synthetic glycopolymers, as artificial matrices, to examine integrin-mediated epidermal growth factor (EGF) signaling in primary hepatocyte cultures. We dispersed hepatocytes on a collagen matrix or on a synthetic glycopolymer matrix and subsequently stimulated them with EGF. Only hepatocytes cultured on collagen proliferated, and we observed significant expression of cyclin B1 in these cells. Pharmacological agents, LY294004 (a phosphatidylinositol [PI] 3-kinase inhibitor) and AG1478 (an EGF kinase receptor inhibitor), blocked hepatocyte proliferation and cyclin B1 expression. In addition, EGF-stimulated hepatocytes formed spheroids, exhibited membrane ruffling, and increased tryptophan 2,3-oxygenase (TO) expression when cultured on glycopolymer matrices. Interestingly, PI 3-kinase inhibition suppressed membrane ruffling, spheroid formation, and TO expression. Taken together, this data suggests PI 3-kinase plays an important role in mediating cross talk between integrin and the EGF signaling pathways in primary hepatocyte cultures.

Hepatocyte behavior on synthetic glycopolymer matrix: inhibitory effect of receptor–ligand binding on hepatocyte spreading

The interaction of carbohydrate-based polymers with asialoglycoprotein receptors (ASGPRs) on the surface of hepatocytes has been used to design hepatocyte adhesion matrices. Therefore, we have characterized the interaction of ASGPR on the surface of hepatocytes with glycopolymer-coated surfaces. Since ASGPRs bound to glycopolymer surfaces escape from internalization and degradation, they were quantified by western blot analysis. The amount of hepatocyte ASGPRs that initially adhered to the glycopolymer surface was proportional to the concentration of the coated glycopolymer. We found that the initial adhesion of hepatocytes to the glycopolymer surface was enhanced by interactions with ASGPR, whereas interactions with ASGPR inhibited the post-adhesion process, a cell adhesion phenomenon that occurs following the initial adhesion. Furthermore, hepatocytes are much more spread on glycopolymer surfaces with lower coating density. Taken together, we suggest that the post-adhesion process triggered hepatocyte spreading on glycopolymer surfaces, and ASGPR–carbohydrate interactions act negatively on the post-adhesion mechanism as well as on hepatocyte spreading on glycopolymer surfaces depending on the density of coated glycopolymers.

Regulation of cell adhesion signaling by synthetic glycopolymer matrix in primary cultured hepatocyte

Control of cell–matrix interactions is a central principle for the design of biomaterial in tissue engineering. In this study, we evaluated a synthetic glycopolymer, which is recognized by the asialoglycoprotein receptor (ASGPR) expressed on the surface of hepatocytes, as an artificial matrix to regulate integrin-mediated signaling. The phosphorylation of focal adhesion kinase was restricted in hepatocytes cultured on the glycopolymer compared with fibronectin. In addition, there was no reorganization of cytoskeleton-related proteins such as actin filaments, microtubules, and vinculin in hepatocytes cultured on the glycopolymer. DNA synthesis and cyclin D1 expression were suppressed in hepatocytes grown on the glycopolymer as compared with those grown on fibronectin and collagen. The data suggest that the glycopolymer will be a good artificial matrix to regulate integrin-mediated signaling and cell growth through the unique ASGPR–carbohydrate interaction.

Probe of specific interaction between a simplified synthetic glycopolymer and erythrocytes as mediated by a glucose transporter (GLUT) on a cell membrane.

In order to develop a biomimetic polymer for cell recognition, we synthesized poly [3-O-(4'-vinylbenzyl)-D-glucose] (PVG), a polystyrene derivative with reduced glucose moiety, and studied the specific interaction of this PVG with erythrocytes, carried by a glucose transporter (GLUT-1), on a cell membrane. To clarify the specific interaction between the PVG and the erythrocytes, fluorescein isothiocyanate- (FITC) labeled polymer was used to prove and visualize the specific interaction. We found that labeled polymer strongly binds to erythrocytes, probably due to the specific interaction mediated by the presence of GLUT-1 on the cell membrane. The fluorescence intensity of PVG on erythrocytes was time and dose dependent. To verify the specific interaction between the PVG and the erythrocytes, cells were pretreated with phloretin, an inhibitor of GLUT-1, before adding the FITC-labeled PVG polymer to the cell-culture medium. This treatment suppressed the interaction of PVG with erythrocytes. A confocal laser microscopic study further confirmed this interaction. The results from this study provide evidence that a biomimetic polymer of PVG interacts with erythrocytes mediated by GLUT-1 on cell membranes.

An ELISA-based assay for detergent-solubilized cellular beta 1,4-galactosyltransferase activity. Use of a polyacrylamide derivative with GlcNAc-beta side chains as a solid phase acceptor substrate.

In our previous paper [Oubihi et al. (1998) Anal. Biochem., 257, 169-175], we have shown that a polyacrylamide-derived synthetic glycopolymer with GlcNAcbeta side chains, termed PAP(GlcNAcbeta), is useful as a solid phase acceptor substrate for the ELISA-based analyses of soluble beta1,4(-)galactosyltransferase (GalT) activity in milk. This method is now used to assay detergent-solubilized cellular GalT. The glycopolymer coated on polystyrene plates was shown to be highly stable against the non-ionic and ionic detergents tested (0 approximately 5% solutions of Triton X-100 and SDS). Such stability made it possible to incubate the ELISA plate with detergent-solubilized GalT and to wash the ELISA plate with SDS solution after the GalT reaction, leading to high accuracy and sensitivity of this assay. The GalT activity was assayed using this method for 1% Triton X-100 extracts of various tissue samples of mice and several cultured cell lines. The results showed that the specific GalT activity of tissue extracts was low in brain and intestine, and high in ovary, muscle, and kidney. As for the cultured cell lines, COS7, COMMA-1D and C2C12 cells showed high specific activity, while CHO and MDCK cells showed low activity. The myoblast C2C12 had a slight increase in GalT activity during starvation-induced cell differentiation. On the other hand, GaIT-I transcript estimated by RT-PCR rather decreased during C2C12 cell differentiation, suggesting a differentiation-dependent switch in GalT isozymes. Taken all together, the ELISA-based assay using PAP(GlcNAcbeta) as a solid phase acceptor substrate was demonstrated to be a useful method for the assay of membrane-bound galactosyltransferases.

Development of an Enzyme-Linked Immunosorbent Assay-Based Method for Measuring Galactosyltransferase Activity Using a Synthetic Glycopolymer Acceptor Substrate

A lectin-assisted enzyme-linked immunosorbent assay (ELISA)-based method using a synthetic glycopolymer as an acceptor substrate was developed for measuring β1,4-galactosyltransferase (GalT) activity. A polyacrylamide derivative having a β-linkedN-acetylglucosamine (GlcNAcβ) moiety on each monomeric unit was synthesized chemically and immobilized on a polystyrene microtiter plate as an acceptor substrate for GalT. After the plate was incubated with bovine GalT, the enzyme reaction product, β-linked Gal residue on the polyacrylamide-bound GlcNAc residue, was detected by usingRicinus communisagglutinin 1 (RCA1), rabbit anti-RCA1 antibody, and a peroxidase-labeled anti-rabbit IgG. The lowest GalT concentration detectable by this method was about 0.5 mU/ml, which is comparable to those by the previously reported ELISA-based assays. The unique property of the glycopolymer, PAP(GlcNAcβ), of binding noncovalently but tightly to the polystyrene microtiter plate allowed the use of this acceptor substrate for the GalT activity measurement even in the presence of 1% Triton CF-54 and X-100. Our system was successfully applied to assess GalT activity in milk of various mammals.