Modulate the phase transition temperature of hydrogels with both thermosensitivity and biodegradability

The synthesis and characterization of thermoresponsive hydrogels on the basis of N-isoproplyarylamide (NIPAAm) and acrylamide (AAm) copolymers crosslinked with a novel biodegradable crosslinker (PEG-co-PLA) were carried out in this study. Swelling measurement results demonstrated that four gels of PNAM5, PNAM10, PNAM12 and PNAM15 are thermoresponsive. The equilibrium swelling ratio and degradation of the hydrogels strongly depend on hydrogels composition. The morphology of the hydrogels was observed by scanning electron microscopy (SEM), and their thermal property was characterized by differential scanning calorimetry (DSC). The results show that the proportion of AAm in the copolymer has notable effect on the low critical solution temperature (LCST) of the hydrogel. When the molar ratio of AAm to NIPAAm was increased from 1:10 to 3:10 the LCST of the copolymer increased from 39.7 to 64.2 °C. The compression modulus of PNAM15 is of the highest among other hydrogels, because PNAM15 hydrogel has a more compact structure.     

Molecular simulation study of the effect of cross-linker on the properties of poly(N-isopropyl acrylamide) hydrogel

Molecular simulation techniques have been utilised to investigate the effect of cross-linker type on the structural and dynamical properties of a temperature-sensitive hydrogel, poly (N-isopropyl acrylamide) (PNIPAM) across its lower critical solution temperature (LCST). PNIPAM exhibits an LCST at ～305 K, above which it collapses and below which it is swollen. Molecular dynamics simulations of PNIPAM hydrogel cross-linked with N, N′-methylene bisacrylamide (BIS) and ethylene glycol dimethacrylate (EGD) cross-linkers were carried out below, at and above its LCST (namely 300, 305 and 310 K, respectively). Structural analysis indicates that the cross-linkers did not affect the temperature of the onset of the LCST, but did affect the degree of swelling and pore size distribution, where the EGD-cross-linked hydrogel exhibited a greater degree of structural change than that of the BIS-cross-linked hydrogel. We believe that this could be attributed to the longer chain length and more flexible nature of the EGD cross-linker compared to the BIS cross-linkers.     

Adsorption of human IgG on to poly(N-isopropylacrylamide)-based polymer particles

Thermosensitive poly(N-isopropylacrylamide)-based polymer particles were synthesised, and screened for the adsorption of human immunoglobulin G (hIgG). At pH 9 the adsorption on microgel particles was strongly affected by temperature, approximately 40 mg hIgG/g support (90% of initial hIgG) being adsorbed at 40°C but only 10% of initial hIgG at 25°C. At pH 5 the maximum adsorbed amount (20 mg hIgG/g support) was similar for both temperatures. The adsorption of hIgG on to charged poly(methyl methacrylate)/poly(N-isopropylacrylamide) core-shell latexes was negligible (5–10 mg hIgG/g support) at the same temperature and pH conditions. The lower adsorption of hIgG onto the core-shell particles is explained by steric interactions due to the small size of the shell.     

Enhanced gene expression through temperature profile-induced variations in molecular architecture of thermoresponsive polymer vectors

BACKGROUND: Successful non-viral gene targeting requires vectors to meet two conflicting needs-strong binding to protect the genetic material during transit and weak binding at the target site to enable release. Responsive polymers could fulfil such requirements through the switching of states, e.g. the chain-extended coil to chain-collapsed globule phase transition that occurs at a lower critical solution temperature (LCST), in order to transport nucleic acid in one polymer state and release it in another. METHODS: The ability of new synthetic polycations based on poly(ethyleneimine) (PEI) with grafted neutral responsive poly(N-isopropylacrylamide) (PNIPAm) chains to condense DNA into particles with architectures varying according to graft polymer LCST was assessed using a combination of fluorescence spectroscopy, dynamic light scattering (DLS), zeta sizing, gel retardation and atomic force microscopy studies. Transfection assays were conducted under experimental conditions wherein the polymer components were able to cycle across their LCST. RESULTS: Two PEI-PNIPAm conjugate polymers with different LCSTs displayed coil-globule transitions when complexed to plasmid DNA, leading to variations in molecular architecture as shown by changes in emission maxima of an environment-sensitive fluorophore attached to the PNIPAm chains. Gel retardation assays demonstrated differences in electrophoretic mobilities of polymer-DNA complexes with temperatures below and above polymer LCSTs. Atomic force micrographs showed changes in the structures of polymer-DNA complexes for a polymer undergoing a phase transition around body temperature but not for the polymer with LCST outside this range. Transfection experiments in C2C12 and COS-7 cells demonstrated that the highest expression of transgene occurred in an assay that involved a 'cold-shock' below polymer LCST during transfection. CONCLUSIONS: Designed changes in thermoresponsive polycation vector configuration via temperature-induced phase transitions enhanced transgene expression. The results indicate that changes in molecular architecture induced by a carefully chosen stimulus during intracellular trafficking can be used to enhance gene delivery.     

New antibody purification procedure using a thermally responsive poly(N-isopropylacrylamide)–dextran derivative conjugate

Through their specificity and affinity, antibodies are useful tools in research and medicine. In this study, we investigated a new type of chromatographic method using a thermosensitive polymer for the purification of antibodies against a dextran derivative (DD), as a model. The thermally reversible soluble–insoluble poly(N-isopropylacrylamide)–dextran derivative conjugate, named poly(NIPAAm)–DD, has been synthesized by conjugating amino-terminated poly(N-isopropylacrylamide) to a DD via ethyl-3-(3-dimethylaminopropyl)-carbodiimide. On one hand, this report describes the two steps of poly(NIPAAm)–DD conjugation and characterization. On the other hand, the poly(NIPAAm)–DD conjugate was used as a tool to purify polyclonal antibodies in serum samples from rabbits subcutaneously immunized with the derivatized dextran. Antibodies were purified and quantified by immunoenzymatic assays. Our results indicate that antibodies recognized both DD and poly(NIPAAm)–DD. In contrast, they did not bind to native poly(NIPAAm) or poly(NIPAAm) conjugated with another anionic dextran. We conclude that the conjugation of a polysaccharide to poly(NIPAAm) leads to an original and efficient chromatographic method to purify antibodies. Moreover, this novel method of purification is rapid, sensitive, inexpensive and could be used to purify various types of antibodies.     

Improved immobilization of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> ATCC 7469 in polyacrylamide gel,preventing cell leakage during lactic acid fermentation

A new procedure for improved immobilization of Lactobacillus rhamnosus ATCC 7469, producing solely l(+)-lactic acid, in polyacrylamide was developed. A series of gels with varied ingredients concentrations and order of addition was prepared and were tested in batch and repeat-batch processes. Our results revealed that the crucial step for successful immobilization was the initial incubation of the cells in pure 10% AA that leads to improved entrapment in the polyacrylamide gel. In contrast, all gels derived from previously prepared stock AA/MBAA released high amount of cells and free biomass was formed. The most efficient immobilization was achieved using gel, containing L. rhamnosus, incubated in 10% AA (acrylamide) and with 1% MBAA (N,N′-methylene-bis-acrylamide) added. This gel possessed optimal permeation characteristics and at the same time, the cells were completely retained in the polymer lattice (0.03 g free biomass/l at 48 h of the batch process). In addition, it yielded highly concentrated lactic acid: the conversion ratio was about 85% without pH-control for initial lactose concentrations of up to 30 g/l. A series of additional immobilization experiments showed the potential of physicochemical interactions between the monomers of acrylamide and the cell surface of L. rhamnosus.     

Nuclear localization signal peptide enhances transfection efficiency and decreases cytotoxicity of poly(agmatine/N,N′-cystamine-bis-acrylamide)/pDNA complexes

At present, nonviral gene vectors develop rapidly, especially cationic polymers. A series of bioreducible poly(amide amine) (PAA) polymers containing guanidino groups have been synthesized by our research team. These novel polymer vectors demonstrated significantly higher transfection efficiency and lower cytotoxicity than polyethylenimine (PEI)—25kDa. However, compared with viral gene vectors, relatively low transfection efficiency, and high cytotoxicity are still critical problems confronting these polymers. In this study, poly(agmatine/N,N′-cystamine-bis-acrylamide) p(AGM-CBA) was selected as a model polymer, nuclear localization signal (NLS) peptide PV7 (PKKKRKV) with good biocompatibility and nuclear localization effect was introduced to investigate its impact on transfection efficiency and cytotoxicity. NLS peptide-mediated in vitro transfection was performed in NIH 3T3 cells by directly incorporating NLS peptide with the complexes of p(AGM-CBA)/pDNA. Meanwhile, the transfection efficiency and cytotoxicity of these complexes were evaluated. The results showed that the transfection efficiency could be increased by 5.7 times under the appropriate proportion, and the cytotoxicity brought by the polymer vector could be significantly reduced.     

Prediction of lower critical solution temperature of N-isopropylacrylamide–acrylic acid copolymer by an artificial neural network model

In this paper, we have investigated the lower critical solution temperature (LCST) of N-isopropylacrylamide–acrylic acid (NIPAAm-AAc) copolymer as a function of chain-transfer agent/initiator mole ratio, acrylic acid content of copolymer, concentration, pH and ionic strength of aqueous copolymer solution. Aqueous solutions with the desired properties were prepared from previously purified polymers, synthesized at 65 °C by solution polymerization using ethanol. The effects of each parameter on the LCST were examined experimentally.In addition, an artificial neural network model that is able to predict the lower cretical solution temperature was develeped. The predictions from this model compare well against both training and test data sets with an average error less than 2.53%.Figure Cross plot of predicted and experimental LCST values for the testing data set.     

Synthesis and characterization of thermosensitive graft copolymer of N-isopropylacrylamide with biodegradable carboxymethylchitosan

A novel thermosensitive and hydrogel was designed and synthesized by graft copolymerization of N-isopropylacrylamide (NIPAAm) with biodegradable carboxymethylchitosan (CMCS). The influence of the content of CMCS grafted on the properties of the resulted hydrogels was examined. The morphology of the hydrogels was observed by scanning electron microscopy (SEM), their thermal property was characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and deswelling/swelling kinetics upon external temperature changes. In comparison with the conventional PNIPAAm hydrogels, the resulted hydrogels have improved thermosensitive properties, including enlarged water content at room temperature and faster deswelling/swelling rate upon heating. The strategy described here presents a potential alternative to the traditional synthesis techniques for thermosensitive hydrogels.     

Highly branched polymers with polymyxin end groups responsive to Pseudomonas aeruginosa

Polymyxin peptide conjugated to the end groups of highly branched poly(N-isopropyl acrylamide) was shown to bind to a Gram negative bacterium, Pseudomonas aeruginosa . The nonbound polymer had a lower critical solution temperature (LCST) above 60 °C. However, binding caused aggregation, which was disrupted on cooling of the bacteria and polymer mixture. The data indicate that polymer binding of bacteria occurred by interaction of the end groups with lipopolysaccharide and that the binding decreased the LCST to below 37 °C. Cooling then progressed the polymer/bacteria aggregate through a bound LCST into an open polymer coil conformation that was not adhesive to P. aeruginosa .     

Thermodynamic analysis of nonelectrolyte sorption in plant cuticles: The effects of concentration and temperature on sorption of 4-nitrophenol

The sorption of 4-nitrophenol (4-NP) in enzymatically isolated cuticles ofLycopersicon esculentum fruits andFicus elastica leaves was studied as a function of temperature and solute concentration. Plots of the concentrations of 4-NP sorbed in the cuticle versus the equilibrium concentrations in the aqueous phase gave linear isotherms at low concentrations that tended to approach plateaus at higher sorbate concentrations ( 10 mmol·kg^-1). At low concentrations of sorbed 4-NP, cuticles have sorptive properties similar to those of organic solvents which are able to form intermolecular hydrogen bonds, while at higher concentrations their solid nature becomes apparent. During sorption of 4-NP the cutin matrix swells and new sorption sites are successively formed. The partition coefficients of 4-NP in the system cuticle/buffer are functions of temperature and concentration. At high sorbate concentrations (approx. 1 mol·kg^-1) they approach a value of 1. Different sorptive properties were observed for the cutin regions normally encrusted with soluble cuticular lipids (SCL) and those without SCL. Increasing temperature augmented the number of sorption sites in the cutin ofLycopersicon while no effect was observed withFicus. The changes of partial molar free energy (G ^o _tr), enthalpy (H ^o _tr), and entropy (S ^o _tr) for the phase transfer of 4-NP also depended on sorbate concentration: H ^o _tr and S ^o _tr were negative and steeply decreased at high sorbate concentrations. This is due to solute-solute interactions replacing solute-cutin interactions at high concentrations resulting in solid precipitates of solute within the cutin matrix. This formation of ordered solid domaines starting from a small number of nonelectrolyte molecules interacting with the cutin is proposed as a model for the intracuticular deposition of SCL.Abbreviations CM cuticular membrane - MX polymer matrix membrane - 4-NP 4-nitrophenol - SCL soluble cuticular lipids     

Simultaneous determination of two human urinary metabolites of N,N-dimethylformamide using gas chromatography–thermionic sensitive detection with mass spectrometric confirmation

Two human urinary metabolites of the industrial solvent N,N-dimethylformamide (DMF), N-hydroxymethyl-N-methylformamide (HMMF) and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), were assayed using a new analytical method (gas chromatography and thermionic sensitive detection). Clean-up of urine samples includes a liquid–liquid extraction step followed by a solid-phase extraction step to separate HMMF and AMCC from other urine components. During clean-up, AMCC is converted into ethyl-N-methylcarbamate (EMC), and during gas chromatography, HMMF is degraded in the injector to N-methylformamide (NMF). All the validation data necessary for a quantitative procedure are given. The method was applied to urine samples from workers exposed to DMF and from the general population. The results were confirmed by mass spectrometric determination. For this purpose a further liquid–liquid extraction step was introduced in the clean-up procedure. Background levels of AMCC in the general population were identified.     

An ionic liquid as reaction media for radiation-induced grafting of thermosensitive poly (N-isopropylacrylamide) onto microcrystalline cellulose

This paper reports a homogeneous modification of microcrystalline cellulose (MCC) in ionic liquids via radiation-induced grafting. Thermosensitive poly (N-isopropylacrylamide) (PNIPAAm) was successfully grafted onto MCC in 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) ionic liquid using γ-ray irradiation technique at room temperature. The grafting yield (GY) increased with dose up to 40 kGy, while decreased slightly with dose rate from 22 to 102 Gy/min. The results of TGA indicated that cellulose grafted PNIPAAm (cellulose-g-PNIPAAm) had higher thermal stability than that of ungrafted regenerated cellulose (reg-cellulose). The crystalline structure of original MCC was largely destroyed during the dissolution process according to the XRD profiles, and grafting PNIPAAm onto cellulose further decreased the intensity of crystallinity. SEM showed that reg-cellulose and cellulose-g-PNIPAAm films displayed dense and homogeneous morphology. Moreover, the resulting cellulose-g-PNIPAAm exhibited obvious thermal sensitivity with a lower critical solution temperature around 35 °C, which was observed from the swelling behavior in water at different temperatures.     

Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis

Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance ¹³C nuclear magnetic resonance spectroscopy identified glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 ± 1.5 to 62.3 ± 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and purified by Ni-NTA His•bind resins. Their substrate specificities and the values of the kinetic parameters were determined by TLC and ¹H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.     

Isolation and characterization of a bacterium that degrades various polyester-based biodegradable plastics

Microorganisms isolated from soil samples were screened for their ability to degrade various biodegradable polyester-based plastics. The most active strain, designated as strain TB-13, was selected as the best strain for degrading these plastics. From its phenotypic and genetic characteristics, strain TB-13 was closely related to Paenibacillus amylolyticus. It could degrade poly(lactic acid), poly(butylene succinate), poly(butylene succinate-co-adipate), poly(caprolactone) and poly(ethylene succinate) but not poly(hydroxybutylate-co-valerate). However, it could not utilize these plastics as sole carbon sources. Both protease and esterase activities, which may be involved in the degradation of plastic, were constitutively detected in the culture broth.     

Coprecipitation of Pseudomonas fluorescens Lipase with Hydrophobic Compounds As an Approach to Its Immobilization for Catalysis in Nonaqueous Media

The precipitation of N-cetylamine, N-cetylacetamide, hexadecane-1,2-diol, cetyl alcohol, and poly(butyl metacrylate) in acetone–water media in the presence of the lipase from Pseudomonas fluorescens was found to be accompanied by the coprecipitation of the enzyme. Within the lyophilized coprecipitates, the lipase exhibits a high catalytic activity and enantioselectivity in the reaction of (1RS)-phenylethanol acetylation with vinyl acetate in t-butyl methyl ether. In order of increasing lipase activity, the coprecipitates can be arranged in the series: cetyl alcohol, poly(butyl metacrylate), hexadecane-1,2-diol, N-cetylamine, and N-cetylacetamide, with the activity 2.5- to 19-fold exceeding the activity of the native enzyme. Immobilization of the lipase on solid supports, such as Celite 545 (physical sorption) and Eupergit C250L (covalent binding), in the presence of hexadecane-1,2-diol was found to increase the esterifying activity of the enzyme.     

Two Critical Factors Affecting the Release of Mitochondrial Cytochrome c as Revealed by Studies Using N,N′-Dicyclohexylcarbodiimide as an Atypical Inducer of Permeability Transition

N,N′-dicyclohexylcarbodiimide (DCCD) was earlier reported to have stimulatory effects on mitochondrial respiration and to induce mitochondrial swelling, when it was added to mitochondrial suspensions. These data seem to imply that DCCD caused the mitochondrial permeability transition (PT), but this possibility had never been investigated. In the present study, effects of DCCD on the mitochondrial structure and function were studied in detail. DCCD was found to induce mitochondrial PT in a cyclosporine A-insensitive manner. Electron microscopic analysis also supported the induction of the mitochondrial PT by DCCD. However, different from many other PT inducers, DCCD failed to cause massive release of mitochondrial cytochrome c. To understand the relationship between the induction of mitochondrial PT and the release of mitochondrial cytochrome c, we compared the actions of DCCD on mitochondrial structure and function with those of Ca²⁺, known as an ordinary PT inducer. As a result, two parameters considered to be critical for controlling the release of mitochondrial cytochrome c on the induction of PT were mitochondrial volume and the velocity of mitochondrial oxygen consumption.     

Crosslinked Poly(tetrahydrofuran) as a Loosely Coordinating Polymer Electrolyte

Solid polymer electrolytes (SPEs) promise to improve the safety and performance of lithium ion batteries (LIBs). However, the low ionic conductivity and transference number of conventional poly(ethylene oxide) (PEO)‐based SPEs preclude their widespread implementation. Herein, crosslinked poly(tetrahydrofuran) (xPTHF) is introduced as a promising polymer matrix for “beyond PEO” SPEs. The crosslinking procedure creates thermally stable, mechanically robust membranes for use in LIBs. Molecular dynamics and density functional theory (DFT) simulations accompanied by ⁷Li NMR measurements show that the lower spatial concentration of oxygen atoms in the xPTHF backbone leads to loosened O–Li⁺ coordination. This weakened interaction enhances ion transport; xPTHF has a high lithium transference number of 0.53 and higher lithium conductivity than a xPEO SPE of the same length at room temperature. It is demonstrated that organic additives further weaken the O–Li⁺ interaction, enabling room temperature ionic conductivity of 1.2 × 10⁻⁴ S cm⁻¹ with 18 wt% N,N‐dimethylformamide in xPTHF. In a solid‐state LIB application, neat xPTHF SPEs cycle with near theoretical capacity for 100 cycles at 70 °C, with rate capability up to 1 C. The plasticized xPTHF SPEs operate at room temperature while maintaining respectable rate capability and capacity. The novel PTHF system demonstrated here represents an exciting platform for future studies involving SPEs.     

Enhancement of the adhesion of fibroblasts by peptide containing an Arg-Gly-Asp sequence with poly(ethylene glycol) into a thermo-reversible hydrogel as a synthetic extracellular matrix

In an effort to regulate the behavior of mammalian cell entrapped in a gel, the gels were functionalized with the putative cell-binding (-Arg-Gly-Asp-) (RGD) domain. The adhesion molecules composed of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides and the cell recognition ligands were inculcated into the thermo-reversible hydrogel composed of N-isopropylacrylamide, with a small amount of succinyl poly(ethylene glycol) (PEG) acrylate (MW 2000) used as the biomimetic extracellular matrix (ECM). The GRGDS-containing p(NiPAAm-co-PEG) copolymer gel was examined in vitro for its ability to promote cell spreading and to increase the viability of the cells by introducing PEG spacers. ECM poorly adhered to hydrogel lacking adhesion molecules permitting only a 20% spread of the seeded cells after 10 days. When the PEG spacer arms, which were immobilized by a peptide linkage, had been integrated into the hydrogel, the conjugation of RGD improved cell spreading by 600% in a 10-day trial.     

Preparation of oligosaccharide-polyacrylamide conjugates and their use as antigens in enzyme immunoassay (EIA)

Oligosaccharides derived fromSalmonella lipopolysaccharides or from human milk were converted to theirN-acetyl-N-(4-acrylamidophenyl)-1-amino-1-deoxyalditol derivatives. These derivatives were copolymerized with acrylamide to give linear, water-soluble polymers, which were used as coating antigens in EIA assays.