Ion-exchange macroporous hydrophilic gel monolith with grafted polymer brushes

The grafting of functional polymers to the pore surface of macroporous monolithic polyacrylamide cryogels was found to be an efficient and convenient method for the preparation of macroporous polyacrylamide gels, so-called cryogels (pAAm cryogels), with both controlled extent of functional group incorporated and with tailored surface chemistries. Anion-exchange polymer chains of poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) and poly([2-(methacryloyloxy)ethyl]-trimethylammonium chloride) (pMETA), and cation-exchange polymer chains of polyacrylate have been grafted onto pAAm cryogels using potassium diperiodatocuprate as initiator. It was possible to achieve the ion-exchange capacity up to 0.2-0.5 mmol/ml. The graft polymerization did not alter the macroporous structure of the pAAm cryogel, however the flow rate of solutes through the cryogel matrix decreased with increase in the density of polymer grafted. The sorption of low-molecular-weight (metal ion, dye) and high-molecular-weight (protein) substances on the grafted monolithic pAAm column has been studied. The results indicate that a 'tentacle'-type binding of protein to grafted polymer depended on the architecture of the grafted polymer layer and took place after a certain degree of grafting has been reached. The binding of proteins by tentacle-like polymer chains allowed for increasing the binding capacity for proteins on the grafted pAAm cryogels up to 6-12 mg/ml.     

Facilitation of gene transfection with well-defined degradable comb-shaped poly(glycidyl methacrylate) derivative vectors

Recently, we reported that ethanolamine (EA)-functionalized poly(glycidyl methacrylate) (PGMA) vectors (PGEAs) can produce good transfection efficiency, while exhibiting very low toxicity. Further improvement in degradability and transfection efficiency of the PGEA vectors will facilitate their application in gene therapy. Comb-shaped cationic copolymers have been of interest and importance as nonviral gene carriers. Herein, the degradable high-molecular-weight comb-shaped PGEA vectors (c-PGEAs) composed of the low-molecular-weight PGEA backbone and side chains were prepared by a combination of atom transfer radical polymerization (ATRP) and ring-opening reactions. The PGEA side chains were linked with the PGEA backbones via hydrolyzable ester bonds. Such comb-shaped c-PGEA vectors possessed the degradability, good pDNA condensation ability, low cytotoxicity, and good buffering capacity. More importantly, the comb-shaped c-PGEA vectors could enhance the gene expression levels. Moreover, the PGEA side chains of c-PGEA could also be copolymerized with some poly(poly(ethylene glycol)ethyl ether methacrylate) species to further improve the gene delivery system.     

Synthesis, characterization, and evaluation as transfection reagents of ampholytic star copolymers: effect of star architecture

Five star polymers based on the positively ionizable hydrophilic 2-(dimethylamino)ethyl methacrylate (DMAEMA) and the hydrophobic but hydrolyzable tetrahydropyranyl methacrylate (THPMA) were prepared by group-transfer polymerization (GTP) using ethylene glycol dimethacrylate (EGDMA) as the coupling agent. In particular, four isomeric star copolymers (one heteroarm, two star block, and the statistical star), all with a 3:1 DMAEMA:THPMA molar ratio, plus one star homopolymer of DMAEMA, with degrees of polymerization of the arms equal to 15, were synthesized. After star polymer preparation and preliminary characterization, the THPMA units were hydrolyzed to negatively ionizable hydrophilic methacrylic acid (MAA) untis, thus yielding star polyampholytes. All the star polyampholytes as well as the commercially available transfection reagent SuperFect were evaluated for their ability to transfect human cervical HeLa cancer cells with the modified plasmid pRLSV40 bearing the enhanced green fluorescent protein (EGFP) as the reporter gene. The transfection efficiency was affected by star architecture. The DMAEMA15-star-MAA5 polyampholyte presented the highest transfection efficiency of all the star polymers tested but lower than that of SuperFect at its optimum conditions. All four star copolymers showed decreased toxicity compared to the DMAEMA star homopolymer for the same amounts of star polymer tested and also compared to the SuperFect at its optimum conditions.     

Design and biophysical characterization of bioresponsive degradable poly(dimethylaminoethyl methacrylate) based polymers for in vitro DNA transfection

Water-soluble, degradable polymers based on poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) with low cytotoxicity and good p-DNA transfection efficiency are highlighted in this article. To solve the nondegradability issue of PDMAEMA, new polymers based on DMAEMA and 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) for gene transfection were synthesized. A poly(ethylene oxide) (PEO) azo-initiator was used as free-radical initiator. PEGylation was performed to improve water solubility and to reduce cytotoxicity of the polymers. The resulting polymers contain hydrolyzable ester linkages in the backbone and were soluble in water even with very high amounts of ester linkages. These degradable copolymers showed significantly less toxicity with a MTT assay using L929 cell lines and demonstrated promising DNA transfection efficiency when compared with the gold standard poly(ethyleneimine). Bioresponsive properties of the corresponding quaternized DMAEMA based degradable polymers were also studied. Although the quaternized DMAEMA copolymers showed enhanced water solubility, they were inferior in gene transfection and toxicity as compared to the unquaternized copolymers.     

Pectic polysaccharides in carrot cells growing in suspension culture

Pectic polysaccharides in the cell wall of suspension-cultured carrot cells (Daucus carota L.) were fractionated into high- and low-molecular-weight components by molecular-sieve chromatography with a Sepharose 4B column. During the phase of cell-wall expansion, the relative content of low-molecular-weight polymers rapidly increased. Electrophoretic analyses of these fractions showed that the high-molecular-weight components were largely composed of neutral and weakly acidic polymers while the low-molecular-weight fraction contained, in addition to neutral polymers, strongly acidic polyuronides in which the content of neutral sugars was very small. The accumulation of a large amount of the strongly acidic polyuronides occurred in a late stage of cell-wall growth, concomitant with a marked decrease in the high-molecular-weight components.Abbreviation MW molecular weight     

Biosynthesis of cell wall peptidoglycan and polysaccharide antigens by protoplasts of type III group B Streptococcus.

The formation of a nascent peptidoglycan-group-specific antigen of type III group B Streptococcus at the cell membrane level was demonstrated with an M-1 mutanolysin-prepared protoplast system. Protoplasts of group B streptococci in suitably stabilized medium (20% sucrose) readily incorporated [3H]acetate into cell surface macromolecules. Four major polysaccharides were isolated from the protoplast cultural supernatant fluid: the peptidoglycan group-specific antigen polymer, the group B-specific antigen, and the low-molecular-weight and high-molecular-weight forms of the type III polysaccharide antigen. Biosynthesis of all four polymers was not affected by the action of chloramphenicol, indicating protein synthesis was not required for the production of polysaccharide in this system. However, all but the low-molecular-weight type III antigen were inhibited by the action of bacitracin, suggesting that three of the polymers share a common synthesis-assembly site in the membrane. Attachment of the high-molecular-weight antigen to the nascent peptidoglycan-group B antigen complex did not occur in the protoplast system, suggesting that a more complex cell wall matrix may be necessary before linkage of the high-molecular-weight antigen takes place.     

Structure-activity relationships of water-soluble cationic methacrylate/methacrylamide polymers for nonviral gene delivery.

A number of water-soluble cationic carriers was evaluated as transfectant. Almost all studied cationic methacrylate/methacrylamide polymers were able to condense the structure of plasmid DNA, yielding polymer/plasmid complexes (polyplexes) with a size of 0.1-0.3 micron and a slightly positive zeta-potential, which can be taken up by cells, e.g., via endocytosis. However, the transfection efficiency and the cytotoxicity of the polymers differed widely: the highest transfection efficiency and cytotoxicity were observed for poly[2-(dimethylamino)ethyl methacrylate], p(DMAEMA). Assuming that polyplexes enter cells via endocytosis, p(DMAEMA) apparently has advantageous properties to escape the endosome. A possible explanation is that, due to its average pK(a) value of 7.5, p(DMAEMA) is partially protonated at physiological pH and might behave as a proton sponge. This might cause a disruption of the endosome, which results in the release of both the polyplexes and cytotoxic endosomal/lysosomal enzymes into the cytosol. On the other hand, the analogues of p(DMAEMA) studied here have a higher average pKa value and have, consequently, a higher degree of protonation and a lower buffering capacity. This might be associated with a lower tendency to destabilize the endosome, resulting in both a lower transfection efficiency and a lower cytotoxicity. Furthermore, molecular modeling showed that, of all studied polymers, p(DMAEMA) has the lowest number of interactions with DNA. We therefore hypothesized that the superior transfection efficiency of p(DMAEMA) containing polyplexes can be ascribed to an intrinsic property of p(DMAEMA) to destabilize endosomes combined with an easy dissociation of the polyplex once present in the cytosol and/or the nucleus.     

Comb polymers prepared by ATRP from hydroxypropyl cellulose

Hydroxypropyl cellulose (HPC) was used as a core molecule for controlled grafting of monomers by ATRP, the aim being to produce densely grafted comb polymers. HPC was either allowed to react with an ATRP initiator or the first generation initiator-functionalized 2,2-bis(methylol)propionic acid dendron to create macroinitiators having high degrees of functionality. The macroinitiators were then "grafted from" using ATRP of methyl methacrylate (MMA) or hexadecyl methacrylate. Block copolymers were obtained by chain extending PMMA-grafted HPCs via the ATRP of tert-butyl acrylate. Subsequent selective acidolysis of the tert-butyl ester moieties was performed to form a block of poly(acrylic acid) resulting in amphiphilic block copolymer grafts. The graft copolymers were characterized by 1H NMR and FT-IR spectroscopies, DSC, TGA, rheological measurements, DLS, and tapping mode AFM on samples spin coated upon mica. It was found that the comb (co)polymers were in the nanometer size range and that the dendronization had an interesting effect on the rheological properties.     

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.     

Endosomal escape of polymeric gene delivery complexes is not always enhanced by polymers buffering at low pH

One of the crucial steps in gene delivery with cationic polymers is the escape of the polymer/DNA complexes ("polyplexes") from the endosome. A possible way to enhance endosomal escape is the use of cationic polymers with a pKa around or slightly below physiological pH ("proton sponge"). We synthesized a new polymer with two tertiary amine groups in each monomeric unit [poly(2-methyl-acrylic acid 2-[(2-(dimethylamino)-ethyl)-methyl-amino]-ethyl ester), abbreviated as pDAMA]. One pKa of the monomer is approximately 9, providing cationic charge at physiological pH, and thus DNA binding properties, the other is approximately 5 and provides endosomal buffering capacity. Using dynamic light scattering and zeta potential measurements, it was shown that pDAMA is able to condense DNA in small particles with a surface charge depending on the polymer/DNA ratio. pDAMA has a substantial lower toxicity than other polymeric transfectants, but in vitro, the transfection activity of the pDAMA-based polyplexes was very low. The addition of a membrane disruptive peptide to pDAMA-based polyplexes considerably increased the transfection efficiency without adversely affecting the cytotoxicity of the system. This indicates that the pDAMA-based polyplexes alone are not able to mediate escape from the endosomes via the proton sponge mechanism. Our observations imply that the proton sponge hypothesis is not generally applicable for polymers with buffering capacity at low pH and gives rise to a reconsideration of this hypothesis.     

Changes in non-cellulosic cell-wall polysaccharides during the growth of carrot cells in suspension cultures

The cell-wall composition of carrot (Daucus carota L.) cells has been studied during their growth in suspension culture. Pectic and hemicellulosic polymers were fractionated according to molecular size by a Sepharose 4B column. Polyuronides in the pectic fraction were resolved into high- and low-molecular-weight components. The low-molecular-weight polyuronides were relatively free of neutral sugars and showed a marked increase during the growth of the cell wall. Hemicellulosic polysaccharides were of disperse molecular size. As cell expansion proceeded, the contents of glucose and xylose in the high-molecular-weight region increased while those in the low-molecular-weight fraction decreased. Removal of auxin from the medium apparently caused degradation of high-molecular-weight polymers in both the pectic and hemicellulosic fractions.     

Physiological and chemical investigations into microbial degradation of synthetic Poly(cis-1,4-isoprene)

Streptomyces coelicolor 1A and Pseudomonas citronellolis were able to degrade synthetic high-molecular-weight poly(cis-1,4-isoprene) and vulcanized natural rubber. Growth on the polymers was poor but significantly greater than that of the nondegrading strain Streptomyces lividans 1326 (control). Measurement of the molecular weight distribution of the polymer before and after degradation showed a time-dependent increase in low-molecular-weight polymer molecules for S. coelicolor 1A and P. citronellolis, whereas the molecular weight distribution for the control (S. lividans 1326) remained almost constant. Three degradation products were isolated from the culture fluid of S. coelicolor 1A grown on vulcanized rubber and were identified as (6Z)-2,6-dimethyl-10-oxo-undec-6-enoic acid, (5Z)-6-methyl-undec-5-ene-2,9-dione, and (5Z,9Z)-6, 10-dimethyl-pentadec-5,9-diene-2,13-dione. An oxidative pathway from poly(cis-1,4-isoprene) to methyl-branched diketones is proposed. It includes (i) oxidation of an aldehyde intermediate to a carboxylic acid, (ii) one cycle of beta-oxidation, (iii) oxidation of the conjugated double bond resulting in a beta-keto acid, and (iv) decarboxylation.     

Fluorescence fluctuation analysis for the study of interactions between oligonucleotides and polycationic polymers

The interactions between a cationic polymer, poly(2-dimethylamino)ethyl methacrylate (pDMAEMA), and negatively charged rhodamine-labeled 25-mer phosphodiester oligonucleotides (Rh-ONs) were studied by fluorescence fluctuation spectroscopy and other techniques. The composition of the pDMAEMA/Rh-ON complexes was investigated as a function of the charge ratio (+/-) by increasing the pDMAEMA concentration and keeping the Rh-ON concentration constant. We applied two different methods for analyzing the fluorescence fluctuation profiles of the pDMAEMA/Rh-ON complexes, which depended on their composition. First, we analyzed the data with the photon counting histogram (PCH) technique, which determines the molecular brightness and the concentration of fluorophores (Chen et al, 1999). A particular challenge for the data analysis is the occurrence of sudden fluorescence bursts in the fluorescence fluctuation profiles, which are linked to the appearance of multimolecular complexes (i. e. when several Rh-ONs were present in one complex). A quantitative interpretation of the analysis for the complexes remains challenging and is connected to the rarity of the fluorescent bursts, which do not provide sufficient data statistics. To specifically address the problem of the fluorescent bursts we employed a method described by Van Craenenbroeck et al. (1999). This method, applicable only when data were integrated over much longer time bins, allowed us to estimate the number of fluorescence bursts which could be considered as a relative measure of the amount of multimolecular complexes present. When monomolecular complexes were formed, i. e. at high values of the charge ratio, highly intense fluorescence peaks were not present and the interpretation of the PCH analysis was more straightforward. The molecular brightness of the species (epsilon), as revealed from PCH analysis, was greater than epsilon for the free Rh-ONs, indicating that the Rh-ONs were attached to pDMAEMA chains.     

Synthesis of biocompatible PEG-Based star polymers with cationic and degradable core for siRNA delivery

Star polymers with poly(ethylene glycol) (PEG) arms and a degradable cationic core were synthesized by the atom transfer radical copolymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate macromonomer (PEGMA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), and a disulfide dimethacrylate (cross-linker, SS) via an "arm-first" approach. The star polymers had a diameter ~15 nm and were degraded under redox conditions by glutathione treatment into individual polymeric chains due to cleavage of the disulfide cross-linker, as confirmed by dynamic light scattering. The star polymers were cultured with mouse calvarial preosteoblast-like cells, embryonic day 1, subclone 4 (MC3T3-E1.4) to determine biocompatibility. Data suggest star polymers were biocompatible, with ≥ 80% cell viability after 48 h of incubation even at high concentration (800 μg/mL). Zeta potential values varied with N/P ratio confirming complexation with siRNA. Successful cellular uptake of the star polymers in MC3T3-E1.4 cells was observed by confocal microscopy and flow cytometry after 24 h of incubation.     

Thermoresponsive poly(N-isopropylacrylamide)-based block copolymer coating for optimizing cell sheet fabrication

Thermoresponsive surfaces are prepared via a spin-coating method with a block copolymer consisting of poly(N-isopropylacrylamide) (PIPAAm) and poly(butyl methacrylate) (PBMA) on polystyrene surfaces. The PBMA block suppresses the removal of deposited PIPAAm-based polymers from the surface. The polymer coating affects the temperature-dependent cellular behavior of the surfaces with respect to protein adsorption. By adjusting layer thicknesses, PBMA-b-PIPAAm-coated surfaces are optimized to regulate the adhesion/detachment of cells by temperature changes. Thus, thermoresponsive polymer-coated surfaces are able to harvest contiguous cell sheets with their basal extracellular matrix proteins.     

A versatile method for the conjugation of proteins and peptides to poly[2-(dimethylamino)ethyl methacrylate].

Random copolymers of 2-(dimethylamino) ethyl methacrylate (DMAEMA) with aminoethyl methacrylate (AEMA) were synthesized by radical polymerization. The amount of incorporated primary amino groups could be controlled by the feed ratio of AEMA to DMAEMA, and was varied from 2 to 6 mol %. Subsequently, protected thiol groups were introduced in a derivatization step with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and subsequent treatment with dithiothreitol (DTT). The obtained thiolated p(DMAEMA-co-AEMA) was conjugated to transferrin (Tf) or the F(ab') fragment of mAb 323/A3 via a disulfide linkage. Moreover, the maleimide derivative of the nuclear localization signal (NLS) decapeptide Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Glu-Asp-NH(2) was coupled to the thiolated polymer via a thioether linkage. The coupling efficiency, as determined by GPC (Tf), SDS-PAGE [F(ab')], or (1)H NMR (NLS peptide) was 90-95% for the Tf conjugate, and more than 95% for the F(ab') conjugate and the NLS conjugate. The synthetic strategy described in this paper is a universal method for the preparation of conjugates of proteins and peptides with pDMAEMA in particular. This method can possibly be used to synthesize protein-polymethacrylate conjugates in general.     

Novel biotin linker with alkyne and amino groups for chemical labelling of a target protein of a bioactive small molecule

We synthesized a novel linker (1) with biotin, alkyne and amino groups for the identification of target proteins using a small molecule that contains an azide group (azide probe). The alkyne in the linker bound the azide probe via an azide-alkyne Huisgen cycloaddition. A protein cross-linker effectively bound the conjugate of the linker and an azide probe with a target protein. The covalently bound complex was detected by western blotting. Linker 1 was applied to a model system using an abscisic acid receptor, RCAR/PYR/PYL (PYL). Cross-linked complexes of linker 1, the azide probes and the target proteins were successfully visualized by western blotting. This method of target protein identification was more effective than a previously developed method that uses a second linker with biotin, alkyne, and benzophenone (linker 2) that acts to photo-crosslink target proteins. The system developed in this study is a method for identifying the target proteins of small bioactive molecules and is different from photo-affinity labelling.     

Methacrylamide polymers with hydrolysis-sensitive cationic side groups as degradable gene carriers

Water-soluble polymers with hydrolyzable cationic side groups (structure of the monomers are shown in Figure 1) were synthesized and evaluated as DNA delivery systems. The polymers, except for pHPMA-NHEM, were able to condense plasmid DNA into positively charged nanosized particles. The rate of hydrolysis at 37 degrees C and pH 7.4 of the side groups differed widely; the fastest rate of hydrolysis was observed for HPMA-DEAE (half-life of 2 h), while HPMA-DMAPr had the lowest rate of hydrolysis (half-life of 70 h). In line with this, pHPMA-DEAE-based polyplexes showed the fastest destabilization of the polyplexes at 37 degrees C and pH 7.4. Polyplexes based on pHPMA-DEAE, pHPMA-DMAE, and pHPMA-MPPM showed release of intact DNA within 24, 48, and 48 h, respectively, after incubation at 37 degrees C and pH 7.4. PHPMA-DEAE and pHPMA-MPPM based polyplexes showed the highest transfection activity (almost twice as active as pEI). Importantly, the pHPMA-DEAE, pHPMA-MPPM, and pHPMA-BDMPAP polyplexes preserved their transfection activity in the presence of serum proteins. All polymers investigated showed a substantial lower in vitro cytotoxicity than pEI. In conclusion, pHPMA-based polyplexes are an attractive class of biodegradable vectors for nonviral gene delivery.     

Poly(3-guanidinopropyl methacrylate): a novel cationic polymer for gene delivery

A cationic polymethacrylate with a guanidinium side group was designed in order to create a polymer with cell membrane-penetrating properties such as Tat or other arginine-rich peptides. The polymer, poly(3-guanidinopropyl methacrylate), abbreviated as pGuaMA, was synthesized by free radical polymerization. The DNA-condensing properties of pGuaMA (Mw 180 kDa) were investigated via dynamic light scattering and zeta potential measurements, and small, positively charged particles (110 nm, +37 mV) were found. It was shown that polyplexes based on pGuaMA were able to transfect COS-7 cells efficiently in the absence of serum, while under the same conditions poly(arginine) (pArg) polyplexes did not show detectable transfection levels. Addition of a membrane-disrupting peptide, INF 7, derived from the influenza virus, to preformed pGuaMA polyplexes did result in approximately 2 times increased transfection levels. DLS, zeta potential measurements, gel electrophoresis, and ethidium bromide displacement measurements indicated that serum induced aggregation of the polyplexes at high polymer/plasmid ratios, while at low polymer/plasmid ratios the polarity of the polyplexes reversed likely due to adsorption of negatively charged proteins on their surface. Likely, the unfavorable interactions of pGuaMA polyplexes with serum proteins is the reason for the absent transfection activity of these polyplexes in the presence of serum. Confocal laser scanning microscopy indicated cellular internalization via endocytosis of both polyplexes and free polymer. Thus, pGuaMA polyplexes enter cells, as reported for other polyplexes, by endocytosis and not, as hypothesized, via direct membrane passage.