Capillary electrophoresis of complex natural polysaccharides

Complex natural polysaccharides, glycosaminoglycans (GAGs), are a class of ubiquitous macromolecules that exhibit a wide range of biological functions and participate and regulate multiple cellular events and (patho)physiological processes. They are generally present either as free chains (hyaluronic acid and bacterial acidic polysaccharides) or as side chains of proteoglycans (PGs; chondroitin/dermatan sulfate, heparin/heparan sulfate, and keratan sulfate) and are most often found in cell membranes and in the extracellular matrix. The recent emergence of modern analytical tools for their study has produced a virtual explosion in the field of glycomics. CE, due to its high resolving power and sensitivity, has been useful in the analysis of intact GAGs and GAG-derived oligosaccharides and disaccharides affording concentration and structural characterization data essential for understanding the biological functions of GAGs. In this review, novel off-line and on-line CE-MS and MS/MS methods for screening of GAG-derived oligosaccharides and disaccharides will be discussed.     

Preparation and drug controlled-release of polyion complex micelles as drug delivery systems

Block copolymers, poly(N-vinylprrolidone)-block-poly(styrene-alter-maleic anhydride) (PVP-b-PSMA) and poly(N-vinylprrolidone)-block-poly(N,N-dimethylaminoethyl methacrylate) (PVP-b-PDMAEMA), were synthesized by reversible addition- fragmentation chain transfer (RAFT) polymerization. In aqueous media, this a pair of oppositely-charged diblock copolymers could self-assemble into stable and narrow distribution polyion complex micelles (PICMs). Transmission electron micrographs (TEM) and dynamic light scattering (DLS) analysis showed that the micelles to be spherically shaped with mean hydrodynamic diameter around 70 nm. In addition, the PICMs display ability to response to external stimuli. All of theses features are quite feasible for utilizing it as a novel intelligent drug delivery system. In order to assess its application in biomedical area, release profiles of coenzyme A (Co A) from PICMs were studied under both simulated gastric and intestinal pH conditions. The release was much quicker in pH 7.4 buffer than in pH 2.0 solution. Based on these results, these PICMs could be a potential pH-sensitive carrier for colon-specific drug delivery system.     

Thermosensitive polyion complex micelles prepared by self-assembly of two oppositely charged diblock copolymers

Polyion complex (PIC) micelles were spontaneously formed in aqueous solutions through electrostatic interaction between two oppositely charged block copolymers, poly(N-isopropylacrylamide)-b-poly(L-glutamic acid) and poly(N-isopropylacrylamide)-b-poly(L-lysine). Their controlled synthesis was achieved via the ring opening polymerization of N-carboxyanhydrides (NCA), ε-benzyloxycarbonyl-L-lysine (Lys(Z)-NCA) or γ-benzyl-L-glutamate (BLG-NCA) with amino-terminated poly(N-isopropylacrylamide) macroinitiator and the subsequent deprotection reaction. The formation of PIC micelles was confirmed by dynamic light scattering and transmission electron microscopy. Turbidimetric characterization suggested that the formed PIC micelles had a concentration-dependent thermosensitivity and their phase transition behaviors could be easily adjusted either by the block length of coplymers or the concentration of micelles.     

Nanosilver-doped DNA polyion complex membrane for electrochemical immunoassay of carcinoembryonic antigen using nanogold-labeled secondary antibodies

A simple and sensitive electrochemical immunoassay protocol was developed for the detection of carcinoembryonic antigen (CEA) using nanosilver-doped DNA polyion complex membrane (PIC) as sensing interface. To construct such an immunosensor, double-stranded DNA was initially assembled onto the surface of thionine/Nafion-modified screen-printed carbon electrode to adsorb silver ions with positive charges, then silver ions were reduced to nanosilver particles with the aid of NaBH₄, and then anti-CEA antibodies were immobilized on the nanosilver surface. Gold nanoparticles conjugated with horseradish peroxidase-labeled anti-CEA were employed as signal antibodies for the detection of CEA with a sandwich-type assay format. Under optimal conditions, the immunosensor exhibited a dynamic range of 0.03-32 ng mL⁻¹ with a low detection limit of 10 pg mL⁻¹ CEA. Intra- and inter-assay imprecision (CVs) were <9.5% and 6.5%, respectively. The response could remain 90.1% of the original current at 30th day. 50 real samples were evaluated using the immunosensor and the enzyme-linked immunosorbent assay, respectively, and received in accordance with those two methods.     

In situ synthesis of polysaccharide nanoparticles via polyion complex of carboxymethyl cellulose and chitosan

Biocompatible polymer-magnetite hybrid nanoparticles were prepared by means of in situ synthesis of magnetite within polysaccharide hydrogel nanoparticles. Hydrogel nanoparticles were first fabricated by blending high-molecular-weight carboxymethyl cellulose as an anionic polymer, and low-molecular-weight chitosan as a cationic polymer to form polyion complexes (CC particles). These polyion complexes were then chemically crosslinked using genipin, a bio-based cross-linker, to form stable nanoparticles having a semi-IPN structure (CCG particles). Magnetite was lastly synthesized within CCG particles by the coprecipitation method to obtain polymer-magnetite hybrid nanoparticles (CCGM particles). The formations of CC, CCG and CCGM particles were mainly observed by transmittance, absorbance of genipin and TEM, respectively, and their hydrodynamic diameters and zeta-potentials were analyzed. It was confirmed that the hydrodynamic diameters and the zeta-potentials of these particles were significantly influenced by pH of the suspension, which was attributed to the charges of polymers. The diameters of CCGM particles were smaller than 200 nm at any pH conditions, suggesting the possibility to apply them as drug delivery carriers. CCGM particles exhibited the responsiveness to a magnetic field in addition to their high dispersion stability, indicating their potential to be utilized as a biomaterial for hyperthermia.     

Preparation of free-standing films of natural polysaccharides using hot press technique and their surface functionalization with biomimetic apatite

This study demonstrated that gel-like polyion complexes obtained by mixing of aqueous solution of chondroitin sulfate, heparin, and hyaluronic acid with that of chitosan were able to form their free-standing films using hot press treatments. These films, having thicknesses ca. 50 and 100 μm, depending on the spacer thickness, were homogeneous and non-porous at the microscopic level, and were not water-soluble. The present fabrication process required neither cross-coupling agents nor introduction of other functional groups to the polysaccharides. Hydroxyapatite deposition on the film surfaces under body fluid conditions was also achieved.     

Development of a novel analytical method for determination of chondroitin sulfate using an in-capillary enzyme reaction

A novel analytical method for determination of total amount of chondroitin sulfate (CS) based on its conversion to desulfated chondro-disaccharide via an enzyme-catalyzed reaction, was developed. Using the in-capillary enzyme reaction, the method was also applied to the successful construction of an on-line analytical system. Within this system, electrophoretic migration was used to mix zones containing the enzyme mixture (chondroitinase ABC, chondro-4-sulfatase, chondro-6-sulfatase and 2-o-sulfatase) and the substrate (CS). The reaction was then allowed to proceed in the presence of a weak electric field and, finally, the product (desulfated chondro-disaccharide) of enzyme reaction migrated to the detector under the influence of an applied electric field. A polyvinyl alcohol-coated capillary was used to reduce protein adsorption. Desulfated chondro-disaccharide was successfully migrated toward the anode in 10 mM Tris-acetate buffer (pH 7.0) under reversed polarity and detected at 232 nm. The established method was validated and demonstrated to be applicable in the determination of total amount of CS in a commercial ophthalmic solution. No interference from the formulation excipients was observed. Good linearity was obtained, with correlation coefficients above 0.999. Recoveries and precisions ranged from 100.0 to 100.5%, and from 0.2 to 0.6% of the relative standard deviation, respectively. Good agreement was obtained between the established method and traditional photometric method based on carbazole reaction. In this study, application of the method to disaccharide compositional analysis was also performed.     

Preparation of a biodegradable matrix through polyion complex formation by mixing polylactide-based microspheres having oppositely charged surfaces

The aggregation of polyions into microsphere (MS) complexes was studied as a preparative method for the construction of a biodegradable matrix. Aqueous suspensions of polylactide (PLA)-based MSs with positively and negatively charged surfaces, MS(K(3)(4+)-PLA) and MS(E(3)(4-)-PLA), respectively, immediately formed aggregates when mixed. The effects of the stoichiometry of the charged groups on the surfaces of the MSs and the ionic strength of the medium on aggregate formation, as well as the degradation behavior of the polyion complex (PIC) matrix over time, were investigated.     

Synthesis and characterization of an ultrathin polyion complex membrane containing β-cyclodextrin for separation of organic isomers

An ultrathin polyion complex (PIC) layer containing β-cyclodextrin (β-CD) was formed on the surface of a charged base membrane. Thus, a positively charged copolymer containing β-CD was synthesized by the radical copolymerization of β-CD monomer and allylamine, and was used to modify the surface of a Nafion membrane containing negatively charged fixed ions. Electron spectroscopy for chemical analysis (ESCA) confirmed the presence of the copolymer on the surface of the Nafion membrane due to the formation of a PIC layer. The ability of the membrane to separate organic isomer mixtures was tested by the pervaporation technique, using butanol isomer mixtures as the feed model. The membrane exhibited a good selectivity toward butanol isomers, indicating the effectiveness of β-CD as a selective fixed carrier for the isomers.     

角膜内皮细胞载体膜片的性质研究及移植试验

利用溶剂浇铸/颗粒沥滤技术制备出壳聚糖和硫酸软骨素共混膜, 研究了共混膜的透光性、 表面结构、 红外图谱、 X-射线衍射图谱以及生物相容性和生物降解性. 结果表明, 共混膜透明度高, 结构均匀, 孔隙大小合适, 壳聚糖分子与硫酸软骨素达到分子水平分散, 且分子间存在较强的相互作用, 共混膜具有良好的生物相容性和生物降解性. 以共混膜为载体培养兔角膜内皮细胞, 结果表明, 共混膜非常适合角膜细胞贴附生长, 10 d内长成良好单层细胞. 将载有角膜内皮细胞的膜片植入内皮缺损兔眼中, 在56 d内术眼基本保持透明, 之后角膜水肿, 逐渐模糊并出现新生血管, 在230 d时术眼新生血管基本消失, 角膜恢复透明.     

Enzyme-catalyzed synthesis of natural and unnatural polysaccharides

Synthesis of natural and unnatural polysaccharide was achieved via “enzymatic polymerization” by utilizing a glycoside hydrolase as catalyst. Particularly, hyaluronan, chondroitin, and their derivatives belonging to glycosaminoglycans have been prepared using sugar oxazoline monomers designed on the basis of the concept “transition-state analogue substrate”. The oxazoline derivatives of N-acetylhyalobiuronate [GlcAβ(1→3)GlcNAc] and N-acetylchondrosine [GlcAβ(1→3)GalNAc], which have the repeating disaccharide structures of hyaluronan and chondroitin, respectively, were successfully polymerized by the catalysis of hyaluronidase, giving rise to synthetic hyaluronan and chondroitin. Their 2-substituted oxazoline derivatives were also polymerized to the corresponding N-acylated hyaluronan and chondroitin derivatives. Furthermore, N-acetylchondrosine oxazoline derivatives sulfated at the C4, the C6, and both the C4 and C6 of the GalNAc unit were catalyzed by hyaluronidase; the monomer sulfated at the C4 was polymerized to chondroitin 4-sulfate with well-defined structure, whereas the other two monomers were exclusively hydrolyzed to the corresponding disaccharides. These different kinds of natural and unnatural polysaccharides having relatively high molecular weights were produced in all cases by the catalysis of hyaluronidase. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 5014–5027, 2006     

Response behavior of sodium-selective electrodes modified by surface attachment of the anticoagulant polysaccharides heparin and chondroitin sulfate

Two different polysaccharides with anticoagulant activities, heparin and chondroitin sulfate, were used to modify the surface of sodium-selective electrodes based on asymmetric cellulose triacetate (CTA) membranes. The membranes were formulated with sodium ionophore X, anionic additive, and o-nitrophenyl octyl ether. The response behavior of the surface-modified sodium electrodes was compared with that of control CTA, as well as poly(vinyl chloride) (PVC)-based sodium-selective electrodes. It was found that the selectivity coefficients obtained with the surface modified CTA membrane electrodes were slightly higher than those of the control, but in the case of heparin-modified electrodes they still met the requirements for analysis of sodium in physiological fluids within an error of <1%; the corresponding error for chondroitin sulfate-modified electrodes was also <1% except for the case of potassium ion in which the error was 1.3%. Likewise, it was found that other response characteristics, such as detection limit, linear range, slope of the response plot, selectivity pattern, and response time were comparable in both the control and the polysaccharide-modified electrodes. Therefore, the surface modification does not significantly alter the response behavior of the sensors.     

Polyelectrolyte complex of chondroitin sulfate and peptide with lower pI value in poly(lactide-co-glycolide) microsphere for stability and controlled release

A polyelectrolyte complex between a therapeutic peptide and chargeable polymer was applied to prevent peptide denaturation in poly(lactide-co-glycolide) (PLGA) microspheres. Chondroitin sulfate A (CsA) was employed as a polymeric additive for the formation of an ionic complex with insulin (InS). The complex prepared at pH 3.0 evidenced a nano-size in the range of 100–400 nm with a mono distribution. The stability of InS in the complex in an organic/water (O/W) interface was verified via RP-HPLC. The insulin in the complex evidenced a retention time almost identical to native InS, whereas free insulin did not evidence such a retention time. On the basis of these studies, PLGA microspheres including a complex with various CsA/InS ratios were prepared via a double-emulsion method (PLGA/CsA MS). InS loading efficiency in the system is higher than that of the microspheres without CsA. The system evidenced a lower initial burst and, following the initial burst, continuous release kinetics for 30 days. Circular dichroism (CD) spectra demonstrated that the insulin in PLGA/CsA MS is more stable than the PLGA-only microspheres (PLGA/only MS) for 20 days. These results indicate that the complex system with CsA is useful for the long-term delivery of peptides with lower pI values.     

Fabrication and characterization of poly(vinyl alcohol)/chitosan hydrogel thin films via UV irradiation

A series of poly(vinyl alcohol)/chitosan (PVA/CTS) hydrogel thin films were prepared via ultraviolet (UV) irradiation, with acrylic acid (AA) monomer added as a crosslinker without the addition of any other photo-initiator. The swelling behaviors, intermolecular chemical bonds, molecular structures, thermal behaviors, degrees of crystallinity, morphologies of the surfaces and internal structure, and their relationship to the AA content were characterized using Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Poly(acrylic acid) (PAA) and its chemical crosslinks formed in hydrogel films via free-radical reactions were confirmed using FTIR and DSC analyses. The XRD patterns indicated that the degree of crystallinity of the hydrogel films decreased as the PAA content was increased. SEM micrographs showed that a uniform interconnected pore structure was formed through the entire hydrogel structure, and a gradient in the crosslinking density through the film thickness was observed to result from extended irradiation times. The swelling behaviors revealed that the formation of PAA and its crosslinking in the hydrogel thin films improved the pH stability and controlled the degree of swelling while retaining a high swelling rate. The successful formation of chemical crosslinking without any specific photo-initiator improves the natural characteristics of CTS and PVA and imparts the resulting PVA/CTS hydrogel thin films with properties that make them very promising in biomedical applications.     

Synthesis of cationic water‐soluble copolymers and hydrogels based on [2‐(methacryloyloxy)ethyl]trimethylammonium chloride and 2‐hydroxyethylacrylate and their complex formation with poly(acrylic acid)

Water‐soluble cationic copolymers and hydrogels were synthesized by radical copolymerization of [2‐(methacryloyloxy)ethyl]trimethylammonium chloride (MADQUAT) and 2‐hydroxyethylacrylate (HEA). The kinetics of copolymerization has been studied and the reactivity ratios were determined. It was found that MADQUAT exhibits higher reactivity in copolymerization. The complexation between linear MADQUAT‐HEA and linear poly(acrylic acid) (PAA) has been studied in aqueous solutions at different pH. It results in the formation of insoluble polyelectrolyte complexes, whose composition and stability to aggregate depends on MADQUAT content in copolymers and pH. The hydrogels were synthesized by three‐dimensional radical copolymerization of MADQUAT and HEA in the presence of a crosslinker. The effects of the feed mixture composition on yield and swelling properties of the hydrogels were studied. The interactions of these hydrogels with linear PAA result in formation of gel–polyelectrolyte complexes and contraction of the samples. It was found that the contraction depends on copolymer composition, PAA molecular weight, and solution pH. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44:845–853, 2006     

Perspectives on composite films of chitosan-based natural products (Ginger,Curcumin, and Cinnamon) as biomaterials for wound dressing

Natural materials are gaining popularity in wound healing and food applications, and they have the potential to alleviate the major environmental problems generated via traditional materials. Biomaterials based on Chitosan incorporated with natural products (Ginger, Curcumin, and Cinnamon) have been fabricated by solvent casting method. The antimicrobial characterization of the prepared samples were also investigated using in vitro antimicrobial studies using Gram-positive microorganism [Micrococcus luteus, Staphylococcus aureus, and Staphylococcus epidermidis], Gram-negative microorganism [Pseudomonas aeruginosa], and pathogenic [Candida albicans]. XRD patterns confirmed the complexation between chitosan and other natural products and this indicate the change in structural of chitosan. The shifting and disappearing of bands in FTIR-ATR and changing in fingerprints in FTIR spectra indicate the homogeneity and interaction between chitosan and the other. Optical properties such as absorption, absorption coefficient, Urbach tail and band gap calculation confirmed that Ginger, Curcumin and Cinnamon interact with chitosan and induces localized state between valence and conduction band. The mechanical properties also studied and revealed that Chitosan/Ginger has the best mechanical characteristic compared the other samples. In addition, the shifting toward higher wavelength for chitosan/Ginger may be utilized to generate electron–hole pairs, which is important for antimicrobial activities, and this confirmed from antimicrobial analysis that Chitosan/Ginger give high antimicrobial activity toward Gram-positive microorganism and suggest it for wound dressing application.     

Ultra-thin emissive molecular devices: polarized emission of Ln(III) complex films

Abstract  On ultra-thin films, Tb(III) shows polarized ff-emission in the visible wavelength region induced by intramolecular energy transfer from the photoexcited organic ligand, such as phenanthroline. Pr(III) causes polarized ππ* emission from the chromophore in the ultra-thin film (Langmuir–Blodgett film). Here, the unique optical properties of molecular devices with lanthanide complexes are summarized. Graphical Abstract        

pH and ionic sensitive chitosan/carboxymethyl chitosan IPN complex films for the controlled release of coenzyme A

pH and ionic sensitive interpenetrating polymer network (IPN) complex films based on chitosan (CS) and carboxymethyl chitosan (CM-CS) were prepared by using glutaraldehyde as crosslinking agent. Its structure was characterized by FT-IR, which indicated that the IPN was formed. The films were studied by swelling, weight loss with time, and release of coenzyme A (CoA). It was found that the IPN films were sensitive to pH and ionic strength of the medium. The cumulative release rate of CoA decreased with CoA loading content, ionic strength or crosslinking agent increasing. The composition of the IPN films and pH of release medium also had significant effect on the release of CoA. The differences in the rates and amounts of released CoA may be attributed to the swelling behavior, the degradation of films, and interaction between drug molecule and polymer matrix. These results suggested CS/CM-CS IPN films could be used as drug delivery carrier.     

Inclusion complexes of the natural product gossypol. Crystal structure of the 2:1 complex of gossypol withm-Xylene

The crystal structure of a 2: 1 inclusion complex of gossypol withm-xylene has been determined by X-ray structure analysis. The crystals of C₃₀H₃₀O₈·0.5C₈ H₁₀ are triclinic, space groupPl^–,a = 8.478(1),b = 14.087(2),c = 14.411(2) Å, = 115,39(1), = 75.11(1), = 86.80(1)°,V = 1475.2(4) ų,Z = 2,D _x = 1.29 g cm^–3,T = 295 K, (CuK ) = 7.01 cm^–1. The structure has been solved by direct methods and refined to the finalR value of 0.079 for 3910 observed reflections. The gossypol molecules are linked by intermolecular hydrogen bonds and form bimolecular layers parallel to the ab plane. Disorderedm-xylene molecules occupy cavities between these layers. All polar groups of the gossypol molecule are packed in the interior of the bilayer while non-polar groups are directed outwards. An analysis of the crystal packing of other inclusion complexes of gossypol shows that such bilayers are formed in four complexes and three of those structures are generically related to each other.Deceased.