Biodegradable interpolyelectrolyte complexes based on methoxy poly(ethylene glycol)-b-poly(alpha,L-glutamic acid) and chitosan

We synthesized methoxy poly(ethylene glycol)-b-poly(alpha,L-glutamic acid) (mPEGGA) diblock copolymer by ring-opening polymerization of N-carboxy anhydride of gamma-benzyl-L-glutamate (NCA) using amino-terminated methoxy polyethylene glycol (mPEG) as macroinitiator. Polyelectrolyte complexation between mPEGGA as neutral-block-polyanion and chitosan (CS) as polycation has been scrutinized in aqueous solution as well as in the solid state. Water-soluble polyelectrolyte complexes (PEC) can be formed only under nonstoichiometric condition while phase separation is observed when approaching 1:1 molar mixing ratio in spite of the existence of hydrophilic mPEG block. This is likely due to mismatch in chain length between polyanion block of the copolymer and the polycation or hydrogen bonding between the components. Hydrodynamic size of primary or soluble PEC is determined to be about 200 nm, which is larger than those reported in some literatures. The increase in polyion chain length of the copolymer leads to the increase in the hydrodynamic size of the water-soluble PEC. Formation of spherical micelles by the mPEGGA/CS complex at nonstoichiometirc condition has been confirmed by the scanning electron microscopy observation and transmission electron microscopy observations. The homopolymer CS experiences attractive interaction with both mPEGA and PGA blocks within the copolymer. Competition of hydrogen bonding and electrostatic force in the system or hydrophilic mPEG segments weakens the electrostatic interaction between the oppositely charged polyions. The existence of hydrogen bonding restrains the mobility of mPEG chains of the copolymer and completely prohibits crystallization of mPEG segments. In vitro culture of human fibroblasts indicates that mPEGGA/CS-based materials have potential in biomedical application, especially in tissue engineering.     

6-Amino-6-deoxy-chitosan. Sequential chemical modifications at the C-6 positions of N-phthaloyl-chitosan and evaluation as a gene carrier

The C-6 positions of chitosan were successively modified in a highly regioselective manner. The starting material, N-phthaloyl-chitosan, was successfully converted into the corresponding 6-deoxy-6-halo derivatives by reaction with N-halosuccinimides and triphenylphosphine in N-methyl-2-pyrrolidone. The resulting chloride and bromide derivatives were then substituted with azido groups by reaction with sodium azide at 120 and 80 degrees C, respectively. The azido groups were then reduced to amines via formation of the triphenylphosphinimine intermediate followed by hydrolysis using aqueous hydrazine, which also led to the removal of the N-phthaloyl groups at the C-2 positions. This sequence gave 6-amino-6-deoxy-chitosan, which, unlike chitosan, is soluble in water at neutral pH. The synthesized 6-amino-6-deoxy-chitosan derivative was evaluated as a gene carrier, and the transfection efficiency for COS-1 cells was shown to be superior to chitosan. In addition, the cytotoxicity was similar to chitosan.     

Methacrylated glycol chitosan as a photopolymerizable biomaterial

Glycol chitosan is a derivative of chitosan that is soluble at neutral pH and possesses potentially useful biological properties. With the goal of obtaining biocompatible hydrogels for use as tissue engineering scaffolds or drug delivery depots, glycol chitosan was converted to a photopolymerizable prepolymer through graft methacrylation using glycidyl methacrylate in aqueous media at pH 9. N-Methacrylation was verified by both (1)H NMR and (13)C NMR. The degree of N-methacrylation, measured via (1)H NMR, was easily varied from 1.5% to approximately 25% by varying the molar ratio of glycidyl methacrylate to glycol chitosan and the reaction time. Using a chondrocyte cell line, the N-methacrylated glycol chitosan was found to be noncytotoxic up to a concentration of 1 mg/mL. The prepolymer was cross-linked in solution using UV light and Irgacure 2959 photoinitiator under various conditions to yield gels of low sol content ( approximately 5%), high equilibrium water content (85-95%), and thicknesses of up to 6 mm. Cross-polarization magic-angle spinning (13)C solid state NMR verified the complete conversion of the double bonds in the gel. Chondrocytes seeded directly onto the gel surface, populated the entirety of the gel and remained viable for up to one week. The hydrogels degraded slowly in vitro in the presence of lysozyme at a rate that increased as the cross-link density of the gels decreased.     

Depolymerization of high-molecular-weight chitosan by the enzyme preparation Celloviridine G20x

A low-molecular-weight water-soluble chitosan was obtained from high-molecular-weight crab chitosan using the enzyme preparation Celloviridine G20x. Optimum conditions for the enzymatic hydrolysis were designed. The reaction should be performed for 4 h in a sodium-acetate buffer (pH 5.2) at 55 degrees C and the enzyme to substrate ratio of 1:400. Fractional extraction of chitosan hydrolysate by aqueous ethanol (ethanol: distilled water) yielded fractions with molecular weights in the range 3.2-26.4 kDa.     

Depolymerization of High-Molecular-Weight Chitosan by the Enzyme Preparation Celloviridine G20x

A low-molecular-weight water-soluble chitosan was obtained from high-molecular-weight crab chitosan using the enzyme preparation Celloviridine G20x. Optimum conditions for enzymatic hydrolysis were designed. The reaction should be performed for 4 h in a sodium-acetate buffer (pH 5.2) at 55°C and an enzyme to substrate ratio of 1 : 400. Fractional extraction of chitosan hydrolysate by aqueous ethanol (ethanol:distilled water) yielded fractions with molecular weights in the range 3.2–26.4 kDa.     

Synthesis of organosoluble chitosan derivatives with polyphenolic side chains

A one-pot synthesis was used to produce chitosan derivatives with polyphenolic side chains via a regioselective phenolic coupling reaction. Under Mannich reaction conditions, treatment of chitosan with formaldehyde and methyl 2,4-dihydroxybenzoate gave N-(2,6-dihydroxy-3-methoxycarbonylphenyl)methylated chitosan in good yield (87%). Formation of a CC bond occurred regioselectively at the C(3) position of methyl 2,4-dihydroxybenzoate. Chitosan derivatives having various phenolic compounds as a side chain were easily synthesized in a similar manner. The chitosan derivatives showed good biodegradability and improved their solubility in methanol (9.8mgmL(-1)) and 2-methoxyethanol (> 10mgmL(-1)). The UV protection provided by the derivatives with phenolic benzophenone side chain was evaluated using UV spectra of polyethylene terephthalate and poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate) films coated with the derivatives and the derivatives absorbed effectively in the UV-A region (<60%). Self-aggregation of the chitosan derivatives with the phenolic side chain was observed by using a fluorescent probe in aqueous solution.     

N-乙酰化反应制备水溶性壳聚糖研究

将高度脱乙酰化的壳聚糖在均相介质中进行N-乙酰化反应,制备水溶性壳聚糖。研究了制备工艺条件对脱乙酰度及水溶性的影响。结果表明,在乙酸—乙醇均相体系中进行乙酰化反应时,壳聚糖与乙酸酐的质量比为1∶0.6,反应温度控制在20℃,反应时间为8 h时,产品的脱乙酰度在50%左右,获得了水溶性良好的N-乙酰化壳聚糖。     

A simple preparation of half N-acetylated chitosan highly soluble in water and aqueous organic solvents

A simple and improved method of preparing highly soluble chitosan (half N-acetylated chitosan) was developed using a series of chitosan samples of low molecular weights, and the solubility of the half N-acetylated chitosan in water and organic solvents was investigated in detail. To reduce the molecular weight, chitosan was treated with NaBO3 under the condition that chitosan was homogeneously dissolved in aqueous acetic acid. Weight-average molecular weights of the obtained chitosan samples were determined using a size-exclusion chromatography system equipped with a low-angle laser light-scattering photometer. Each chitosan sample was then N-acetylated with acetic anhydride under the condition that chitosan was homogeneously dissolved in aqueous acetic acid again. The water solubility of the half N-acetylated chitosan thus prepared increased with decreasing molecular weight. From 1H NMR spectroscopy, it was suggested that the sequence of N-acetylglucosamine and glucosamine residues was random. The solubility of the half N-acetylated chitosan of low molecular weight was rather high even in aqueous dimethylacetamide and dimethylsulfoxide.     

Separation and purification of methoxypoly(ethylene glycol) grafted red blood cells via two-phase partitioning

Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. To attenuate the risk of alloimmunization, we have pioneered the membrane grafting of methoxypoly(ethylene glycol) (mPEG) to produce immunocamouflaged red blood cells (RBC). Grafting of the mPEG was accomplished using cyanuric chloride activated mPEG (CmPEG; M(r) = 5000), benzotriazole carbonate methoxyPEG (BTCmPEG; M(r) = 2000, 5000 or 20000); or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPAmPEG; M(r) = 5000, or 20000). Because of the heterogeneity of grafting, a crucial tool in developing the stealth RBC is an ability to purify the modified RBC from unmodified (immunogenic) donor cells. As demonstrated, a (5, 4) dextran:PEG aqueous two-phase polymer partitioning system cleanly separated the immunologically silent mPEG-grafted human RBC from control or lightly modified cells. Cell mixing experiments employing varying ratios of mPEG-modified and control RBC confirmed the purification efficacy of the phase partitioning system. Proportional changes in PEG-rich phase partitioning were achieved by increasing either the quantity of surface mPEG or the mPEG molecular weight. The biological viability of purified mPEG-RBC (BTCmPEG; [M(r) = 20000) was demonstrated by their normal in vivo survival at immunoprotective grafting concentrations (相似文献   

A novel soft and cotton-like chitosan-sugar nanoscaffold

A novel type of chitosan nanoscaffold with a soft and cotton-like appearance is proposed. The key to success is based on two points: (i) the change in morphology of chitin whisker to chitosan nanoscaffold and (ii) the surface modification of the nanoscaffold chitosan with a sugar unit. Simple deacetylation of chitin whisker gives a colloidal solution of chitosan, of which the chitosan is in a nanoscaled scaffold. Surface functionalization of the chitosan nanoscaffold with lactose or maltose via a heterogeneous system in water at room temperature results in a soft and cotton-like chitosan containing mesopores. As all steps are organic solvent free, this chitosan-sugar nanoscaffold might be a promising material for biopolymer-supported tissue engineering.     

Chemoenzymatic syntheses of amylose-grafted chitin and chitosan

Amylose-grafted chitin and chitosan were synthesized by chemoenzymatic methods according to the following reaction manners. First, maltoheptaose was introduced to chitosan by a reductive amination using sodium cyanotrihydroborate in a mixed solvent of 1.0 mol/L aqueous acetic acid and methanol at room temperature to produce a maltoheptaose-grafted chitosan (1). The functionality of maltoheptaose to chitosan in 1 depended on reaction time. The phosphorylase-catalyzed enzymatic polymerization of R-D-glucose 1-phosphate was then performed from 1 to obtain amylose-grafted chitosan (2). Maltoheptaose-grafted chitin (3) was synthesized by N-acetylation of 1 using acetic anhydride in a mixed solvent of aqueous acetic acid and methanol. Then, synthesis of amylose-grafted chitin (4) was performed by the phosphorylase-catalyzed enzymatic polymerization under conditions the same as those for 2. The average DPs of amylose graft chains in 2 and 4 depended on the feed ratios of R-D-glucose 1-phosphate to maltoheptaose primers in 1 and 3.     

A series of novel chitosan derivatives: Synthesis, characterization and micellar solubilization of paclitaxel

A series of novel chitosan derivatives with octyl, sulfate and polyethylene glycol monomethyl ether (mPEG) groups as hydrophobic and hydrophilic moieties, respectively, were synthesized. These PEGylated amphiphilic chitosan derivatives were characterized with ¹H NMR, ¹³C NMR, FTIR and elemental analysis. And their physical properties were measured by wide angle X-ray diffraction (WAXD) and thermogravimetric analysis (TG). The critical micelle concentrations (CMCs) of the modified chitosans determined by using pyrene as a hydrophobic probe in fluorescence spectroscopy were found to be 0.011–0.079 mg/ml, and the log CMC was linearly relative to four structure parameters, that is the degree of substitution (DS) of chitosan unit, sulfate group, PEG unit and octyl group by mole per kilogram. Paclitaxel, a water-insoluble anticancer drug, was solubilized into the polymeric micelles formed by these derivatives utilizing physical entrapment method, with micellar particle size around 100–130 nm, and the highest paclitaxel concentration of 3.94 mg/ml was found in N-mPEG-N-octyl-O-sulfate chitosan (mPEGOSC) micellar solution, which was much higher than that in water (less than 0.001 mg/ml). Therefore, N-mPEG-N-octyl-O-sulfate chitosan micelles may be useful as a prospective carrier for paclitaxel.     

PEGylation of bovine serum albumin using click chemistry for the application as drug carriers

Monomethyl poly(ethylene glycol) (mPEG)-modified bovine serum albumin (BSA) conjugates (BSA-mPEG) were obtained by the mild Cu(I)-mediated cycloaddition reaction of azided BSA (BSA-N(3) ) and alkyne-terminated mPEG. The structure and characteristics of BSA-mPEG conjugates were thoroughly investigated. There were about two PEG chains conjugated onto each BSA molecule as determined by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) analysis. The intrinsic nonspecific binding ability of BSA was used for adsorption and sustained release of both rifampicn and 5-fluorouracil (5-FU). The helical structures of BSA were preserved to a large extent after modification and drug adsorption on BSA was confirmed via circular dichroism spectroscopy. Drugs adsorbed onto the conjugated formulation to a lesser extent than on BSA due to mPEG modification. The in vitro release of both rifampicin and 5-FU, however, indicated that BSA-mPEG can function as a drug carrier. Overall, the click reaction provided a convenient tool for the pegylation of BSA. The biological activity of the BSA-mPEG conjugates, including the drug transportation capacity and biocompatibility, were largely retained.     

Synthesis and properties of chitosan hydrogels modified with heterocycles

Preparation and properties of chitosan modified with heterocycles in absence or presence of gluteraldehyde as a cross linker is described. New modified chitosan–heterocyclic hydrogels were prepared from chitosan and heterocyclic compounds such as N,N′-biisomaleimide, N,N′-biisophthalimide, and N,N′-phthalimidomaleimide via a crosslinking reaction. The new hydrogels chemical structure was characterized by spectral analysis (IR), X-ray diffraction, thermal gravimetric analysis (TGA), solubility, and swellability in water and different organic solvents. Evaluation of the efficiency of the new hydrogels to uptake copper and cobalt ions from aqueous systems was carried out and promising results were obtained.     

Poly(N-isopropylacrylamide) surface-grafted chitosan membranes as a new substrate for cell sheet engineering and manipulation

The immobilization of poly(N-isopropylacrylamide) (PNIPAAm) on chitosan membranes was performed in order to render membranes with thermo-responsive surface properties. The aim was to create membranes suitable for cell culture and in which confluent cell sheets can be recovered by simply lowering the temperature. The chitosan membranes were immersed in a solution of the monomer that was polymerized via radical initiation. The composition of the polymerization reaction solvent, which was a mixture of a chitosan non-solvent (isopropanol) and a solvent (water), provided a tight control over the chitosan membranes swelling capability. The different swelling ratio, obtained at different solvent composition of the reaction mixture, drives simultaneously the monomer solubility and diffusion into the polymeric matrix, the polymerization reaction rate, as well as the eventual chain transfer to the side substituents of the pyranosyl groups of chitosan. A combined analysis of the modified membranes chemistry by proton nuclear magnetic resonance ((1)H-NMR), Fourier transform spectroscopy with attenuated total reflection (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) showed that it was possible to control the chitosan modification yield and depth in the solvent composition range between 75% and 100% of isopropanol. Plasma treatment was also applied to the original chitosan membranes in order to improve cell adhesion and proliferation. Chitosan membranes, which had been previously subjected to oxygen plasma treatment, were then modified by means of the previously described methodology. A human fetal lung fibroblast cell line was cultured until confluence on the plasma-treated thermo-responsive chitosan membranes and cell sheets were harvested lowering the temperature.     

Synthesis and characterization of poly(ethylene glycol)-insulin conjugates

Human insulin was modified by covalent attachment of short-chain (750 and 2000 Da) methoxypoly (ethylene glycol) (mPEG) to the amino groups of either residue PheB1 or LysB29, resulting in four distinct conjugates: mPEG(750)-PheB1-insulin, mPEG(2000)-PheB1-insulin, mPEG(750)-LysB29-insulin, and mPEG(2000)-LysB29-insulin. Characterization of the conjugates by MALDI-TOF mass spectrometry and N-terminal protein sequence analyses verified that only a single polymer chain (750 or 2000 Da) was attached to the selected residue of interest (PheB1 or LysB29). Equilibrium sedimentation experiments were performed using analytical ultracentrifugation to quantitatively determine the association state(s) of insulin derivatives. In the concentration range studied, all four of the conjugates and Zn-free insulin exist as stable dimers while Zn(2+)-insulin was exclusively hexameric and Lispro was monomeric. In addition, insulin (conjugate) self-association was evaluated by circular dichroism in the near-ultraviolet wavelength range (320-250 nm). This independent method qualitatively suggests that mPEG-insulin conjugates behave similarly to Zn-free insulin in the concentration range studied and complements results from ultracentrifugation studies. The physical stability/resistance to fibrillation of mPEG-insulin conjugates in aqueous solution were assessed. The data proves that mPEG(750 and 2000)-PheB1-insulin conjugates are substantially more stable than controls but the mPEG(750 and 2000)-LysB29-insulin conjugates were only slightly more stable than commercially available preparations. Circular dichroism studies done in the far ultraviolet region confirm insulin's tertiary structure in aqueous solution is essentially conserved after mPEG conjugation. In vivo pharmacodynamic assays reveal that there is no loss in biological activity after conjugation of mPEG(750) to either position on the insulin B-chain. However, attachment of mPEG(2000) decreased the bioactivity of the conjugates to about 85% of Lilly's HumulinR formulation. The characterization presented in this paper provides strong testimony to the fact that attachment of mPEG to specific amino acid residues of insulin's B-chain improves the conjugates' physical stability without appreciable perturbations to its tertiary structure, self-association behavior, or in vivo biological activity.     

Stability improvement of electrospun chitosan nanofibrous membranes in neutral or weak basic aqueous solutions

Further utilization of chitosan nanofibrous membranes that are electrospun from chitosan solutions in trifluoroacetic acid (TFA) with or without dichloromethane (DCM) as the modifying cosolvent is limited by the loss of the fibrous structure as soon as the membranes are in contact with neutral or weak basic aqueous solutions due to complete dissolution of the membranes. Dissolution occurs as a result of the high solubility in these aqueous media of -NH(3)(+)CF(3)COO(-) salt residues that are formed when chitosan is dissolved in TFA. Traditional neutralization with a NaOH aqueous solution only maintained partial fibrous structure. Much improvement in the neutralization method was achieved with the saturated Na(2)CO(3) aqueous solution with an excess amount of Na(2)CO(3)(s) in the solution. We showed that electrospun chitosan nanofibrous membranes, after neutralization in the Na(2)CO(3) aqueous solution, could maintain its fibrous structure even after continuous submersion in phosphate buffer saline (pH = 7.4) or distilled water for 12 weeks.     

Effect of carboxymethylation conditions on the water-binding capacity of chitosan-based superabsorbents

A superabsorbent polymer (SAP) from chitosan was provided via carboxymethylation of chitosan, followed by cross-linking with glutaraldehyde and freeze-drying. This work was focused on an investigation of the effects of monochloroacetic acid (MCAA), sodium hydroxide, and reaction time on preparation of carboxymethyl chitosan (CMCS). The CMCS products were characterized using FTIR spectroscopy, and their degrees of substitution (DS) were measured using conductimetry and FTIR analysis. The highest DS value was obtained when the carboxymethylation reaction was carried out using 1.75 g MCAA and 1.75 g NaOH per g of chitosan in 4 h. The water solubilities of the CMCS products at various pHs were also evaluated, and the results indicated a significant impact of the reaction parameters on the solubility of CMCS. The CMCSs with the highest DS value resulted in SAPs having the highest water-binding capacity (WBC). The WBC of the best SAP measured after 10 min exposure in distilled water, 0.9% NaCl solution, synthetic urine, and artificial blood was 104, 33, 30, and 57 g/g, respectively. The WBC of this SAP at pH 2-9 passed a maximum at pH 6.     

Facile, efficient approach to accomplish tunable chemistries and variable biodistributions for shell cross-linked nanoparticles

The in vivo behavior of shell cross-linked knedel-like (SCK) nanoparticles is shown to be tunable via a straightforward and versatile process that advances SCKs as attractive nanoscale carriers in the field of nanomedicine. Tuning of the pharmacokinetics was accomplished by grafting varied numbers of methoxy-terminated poly(ethylene glycol) (mPEG) chains to the amphiphilic block copolymer precursors, together with chelators for the radioactive tracer and therapeutic agent (64)Cu, followed by self-assembly into block copolymer micelles and chemical cross-linking throughout the shell regions. (64)Cu-radiolabeling was then performed to evaluate the SCKs in vivo by means of biodistribution experiments and positron emission tomography (PET). It was found that the blood retention of PEGylated SCKs could be tuned, depending on the mPEG grafting density and the nanoparticle surface properties. A semiquantitative model of the density of mPEG surface coverage as a function of in vivo behavior was applied to enhance the understanding of this system.     

Successful removal of p-quinone with chitosan in an aqueous phase in relation to degree of deacetylation

Phenol oxidant is successfully removed by using chitosan particles in the aqueous phase. Removal of p-quinone by chitosan from crab shells was investigated kinetically from molecular weight (MW) of chitosan, deacetylation degree (DD) and reaction temperature. The rate constant assuming first-ordered reaction on removal of p-quinone in aqueous phase primarily depended on the MW of chitosan, not on the DD. Quantities of chitosan exceeding 5 x 10(5) MW are able to obtain a sufficiently high rate constant (10(-3) s(-1)). At higher temperatures, higher rate constants were obtained in the entire experimental MW and DD. The activation energy obtained was 43.8 kJ x mol(-1).