Preparation, characterization, and in vitro drug release behavior of biodegradable chitosan-graft-poly(1, 4-dioxan-2-one) copolymer

A novel copolymer of chitosan-g-poly(p-dioxanone) (CGP) was synthesized in bulk by ring-opening polymerization of p-dioxanone (PDO) initiated by the hydroxyl group or amino group of chitosan using SnOct₂ as catalyst. The chemical structure was determined by ¹H NMR. It was found that the feed ratio of chitosan to PDO had a great effect on the degree of polymerization (DP) and the substitution (DS) of PDO. The thermal stability and crystallization behavior of graft copolymer CGP were closely related to the values of DP and DS. When the resulting copolymer was used as Ibuprofen carrier, the release rate of Ibuprofen decreased compared with that of pure chitosan carrier. The drug release behavior was also influenced by the structure of graft copolymers.     

Synthesis and characterization of a brush-like copolymer of polylactide grafted onto chitosan

A brush-like poly(DL)-lactide grafted onto chitosan as the backbone was investigated. The graft copolymerization was carried out with triethylaluminum as catalyst in toluene at 70 degrees C. It was found that a greater lactide content in the feeding ratio results in a higher grafting percentage. FTIR spectrometry, (1)H NMR, DSC scanning, and wide-angle X-ray scattering, respectively, are used to characterize these branch copolymers. A copolymer has a definite melting point when the molar feeding ratio of lactide to chitosan is more than 10:1, and the deltaH of the copolymers increases with the feed ratio of lactide to chitosan in feeding.     

Preparation and characterization of poly(ethylene glycol)-g-chitosan with water- and organosolubility

A new scheme was proposed for synthesizing poly(ethylene glycol)-g-chitosan (PEG-g-CS), where methoxy poly(ethylene glycol) iodide (MPEG-I) (M_n 2000) was used for N-substitution of triphenylmethyl chitosan (TPM-CS) in organic medium. The graft copolymers were obtained by subsequent removal of protecting groups with dichloroacetic acid. By varying PEG-I/TPM-CS feed ratio, the grafting levels (GL) of PEG can be adjusted. The chitosan derivatives were characterized by FTIR, ¹H NMR, ¹³C NMR and DSC. All the copolymers were soluble in water over wide pH range. Furthermore, organosolubility of the hybrids in DMF and DMSO was also achieved when the DS value more than 24%. The lysozyme degradation rate of the copolymers in aqueous neutral medium decreased with the increase of GL value.     

Synthesis and characterization of poly(dimer acid-brassylic acid) copolymer and poly(dimer acid-pentadecandioic acid) copolymer

Poly(dimer acid-brassylic acid) [P(DA-BA)] copolymers and poly(dimer acid-pentadecandioic acid) [P(DA-PA)] copolymers were prepared by melt polycondensation of the corresponding mixed anhydride prepolymers. The copolymers were characterized by Fourier transform infrared (FTIR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), wide angle x-ray powder-diffraction, and thermal gravimetric analysis (TGA). In vitro studies show that all the copolymers are degradable in phosphate buffer at 37 degrees C, and leaving an oily dimer acid residue after hydrolysis for the copolymer with high content of dimer acid. The release profiles of hydrophilic model drug, ciprofloxcin hydrochloride, from the copolymers, follow first-order release kinetics. All the preliminary results suggested that the copolymer might be potentially used as drug delivery devices.     

Preparation and spectroscopic characterization of methoxy poly(ethylene glycol)-grafted water-soluble chitosan

The object of this study was to test the solubility of a methoxy poly(ethylene glycol) (MPEG)-grafted chitosan copolymer in organic solvents and aqueous solution. Water-soluble chitosan with low molecular weight (LMWSC) was used in a PEG-graft copolymerization. The MPEG was conjugated to chitosan using 4-dicyclohexylcarbodimide (DCC), and N-hydroxysuccimide (NHS). Introduction of PEG was confirmed by (1)H and (13)C NMR spectroscopy and FT-IR spectroscopy. The degree of substitution (DS) of MPEG into chitosan was calculated from (1)H NMR data and also by estimating the molecular weight (MW) using gel permeation chromatography (GPC). The DS values obtained from (1)H NMR spectroscopy and GPC were similar, indicating that MPEG-grafted LMWSC was synthesized and properly characterized. Furthermore, the introduction of PEG into chitosan increases the solubility in aqueous solutions over a range of pH values (4.0-11.0) and organic solvents such as DMF, DMSO, ethanol, and acetone.     

（S）-（-）-β-羟基苯丙酸乙酯的微生物法合成

采用酿酒酵母CGMCC No．2266菌体,不对称还原β-羰基苯丙酸乙酯制备光学纯（S）-（-）-β-羟基苯丙酸乙酯。结果表明：采用初始pH为8．0的液体发酵培养基培养的CGMCCNo．2266菌体经过50℃预热处理30min后用于生物转化获得的（S）-（-）-G-羟基苯丙酸乙酯对映体过剩值可以达到100％ee。确定了合成（S）-（-）-β-羟基苯丙酸乙酯的较佳转化条件为pH7．0,温度30℃,转化时间24h,底物浓度为3．63mmol／L,菌体用量为86g/L（干重／反应体积）。以10％葡萄糖为辅助底物,产率比不加辅助底物时提高了75．4％。在最佳转化条件下反应转化率及（S）-（-）-β-羟基苯丙酸乙酯对映体过剩值可分别达到98．4％和100％ee。     

Preparation and PCR-amplification properties of a novel amphiphilic poly(N-vinylpyrrolidone) (PVP) copolymer

A novel amphiphilic copolymer, P(NVP-co-TrpAMT) (9) was prepared, comprising hydrophilic N-vinylpyrrolidone (NVP; 8) and hydrophobic 'N-[(tert-butoxy)carbonyl]tryptophanamido-N'-methacryl thiourea' (TrpAMT; 7) segments. The amphiphilic copolymer 9 was characterized by (1)H-NMR, GPC-MALLS, TEM, and MTT assay. It has a critical micelle concentration (cmc) of 45.7 mg/l in aqueous solution, and good biocompatibility in vivo. According to TEM, the polymer is mostly present as spherical micelles in water, with a diameter of ca. 60-90 nm. In the presence of 0.1 mug/ml of 9, the PCR amplification of the GC-rich beta-actin was efficiently enhanced. Also, the fluorescence intensity of the reporter dye SYBR Green I was increased by 26% at the 14th cycle during real-time PCR of plasmid pUC18 DNA.     

A novel composite nanofiltration (NF) membrane prepared from graft copolymer of trimethylallyl ammonium chloride onto chitosan (GCTACC)/poly(acrylonitrile) (PAN) by epichlorohydrin cross-linking

A novel composite nanofiltration (NF) membrane was prepared by over-coating the PAN ultrafiltration (UF) membrane with a GCTACC thin layer. The effects of membrane preparation techniques and operating conditions on the performance of the composite membrane were studied. The results indicate that a composite NF membrane from 1.0wt% GCTACC casting solution, vaporized for 2h at 50 degrees C, cross-linked for 20h at 50 degrees C and pH approximately 12 with ethanol/epichlorohydrin (50/0.45 wt/wt) had optimum performance. The resultant GCTACC/PAN composite membrane was positively charged. Scanning electron microscopy showed its asymmetric and composite features. At 25 degrees C and 30L/h of cycling flow, the permeability of pure water through this membrane is 6.3L/hm(2)MPa. At 25 degrees C, 1.2MPa and 30L/h of cycling flow, the rejection of 1000mg/L MgCl(2), CaCl(2), MgSO(4), Na(2)SO(4), and NaCl solutions is 0.976, 0.972, 0.897, 0.65, and 0.407, respectively, with fluxes of 6.8, 6.12, 6.12, 5.57, and 5.51L/hm(2), respectively. The order of rejection of different salts follows the decreasing order of MgCl(2), CaCl(2), MgSO(4), NaCl, KCl, Na(2)SO(4), and K(2)SO(4), which reveals the characteristics of the positively charged NF membrane. In addition, the curve for the streaming potential also illustrates the positively charged characteristics of this membrane, with a pressure osmotic coefficient of 11.7mVMPa(-1).     

Preparation and characterization of N-(2-carboxybenzyl)chitosan as a potential pH-sensitive hydrogel for drug delivery

A novel water-soluble chitosan derivative [N-(2-carboxybenzyl)chitosan, CBCS] was synthesized. The chemical structure of CBCS was characterized by FTIR, (1)H NMR and UV spectroscopies. The degree of substitution (DS) of N-2-carboxybenzyl was determined by colloid titration. In different pH buffer solutions, the swelling characteristics of hydrogels based on CBCS (CBCSG) prepared by crosslinking with glutaraldehyde have been studied. Results showed that the swelling ratio (SR) of CBCSG decreased with an increase of the amount of glutaraldehyde, and that CBCSG swelled more significantly in alkaline solution than in acidic medium, showing the lowest SR at pH5.0. The SR of CBCSG increased with the raising of the DS of the N-2-carboxybenzyl group in alkaline solution, but no significant change was observed in an acidic environment. CBCSG showed swelling reversibility when alternately soaked in pH1.0 and 7.4 buffer solutions. Release profiles of fluorouracil (5-FU), a poorly water-soluble drug, from CBCSG were studied under both simulated gastric and intestinal pHconditions. The release was much quicker in pH7.4 buffer than in pH1.0 solution. Results indicated that CBCS could be a potential pH-sensitive carrier for colon-specific drug delivery system.     

Synthesis, characterization and applications of graft copolymer (Chitosan-g-N,N-dimethylacrylamide)

An unreported graft copolymer of N,N-dimethylacrylamide (DMA) with chitosan has been synthesized under nitrogen atmosphere using peroxymonosulphate/mandelic acid redox pair. The effect of reaction conditions on grafting parameters i.e. grafting ratio, efficiency, conversion, add on and homopolymer has been studied. Experimental results show that maximum grafting has been obtained at 1.0 g dm⁻³ concentration of chitosan, 30 × 10⁻² mol dm⁻³ concentration of N,N-dimethylacrylamide and 7.0 × 10⁻³ mol dm⁻³ concentration of hydrogen ion. It has also been observed that grafting ratio, add on, conversion and efficiency increase upto 3.2 × 10⁻³ mol dm⁻³ of mandelic acid, 12.0 × 10⁻³ mol dm⁻³ of potassium peroxymonosulphate, 150 min of time and 40 °C of temperature. Grafted polymer has been characterized by FTIR spectroscopy and thermogravimetric analysis. Water swelling capacity of chitosan-g-N,N-dimethylacrylamide has been determined. It has been observed that the graft copolymer is thermally more stable than parent backbone.     

Preparation and characterization of poly(acrylic acid)-hydroxyethyl cellulose graft copolymer

Poly(acrylic acid) hydroxyethyl cellulose [poly(AA)-HEC] graft copolymer was prepared by polymerizing acrylic acid (AA) with hydroxyethyl cellulose (HEC) using potassium bromate/thiourea dioxide (KBrO(3)/TUD) as redox initiation system. The polymerization reaction was carried out under a variety of conditions including concentrations of AA, KBrO(3) and TUD, material to liquor ratio and polymerization temperature. The polymerization reaction was monitored by withdrawing samples from the reaction medium and measuring the total conversion. The rheological properties of the poly(AA)-HEC graft copolymer were investigated. The total conversion and rheological properties of the graft copolymer depended on the ratio of KBrO(3) to TUD and on acrylic acid concentration as well as temperature and material to liquor ratio. Optimum conditions of the graft copolymer preparation were 30mmol KBrO(3) and 30mmol TUD/100g HEC, 100% AA (based on weight of HEC), duration 2h at temperature 50°C using a material to liquor ratio of 1:10.     

Antialgal activity of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes against the marine alga Ulva

Marine biofouling has detrimental effects on the environment and economy, and current antifouling coatings research is aimed at environmentally benign, non-toxic materials. The possibility of using contact-active coatings is explored, by considering the antialgal activity of cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes. The antialgal activity was investigated via zoospore settlement and sporeling growth assays of the marine algae Ulva linza and U. lactuca. The assay results for PDMAEMA brushes were compared to those for anionic and neutral surfaces. It was found that only PDMAEMA could disrupt zoospores that come into contact with it, and that it also inhibits the subsequent growth of normally settled spores. Based on the spore membrane properties, and characterization of the PDMAEMA brushes over a wide pH range, it is hypothesized that the algicidal mechanisms are similar to the bactericidal mechanisms of cationic polymers, and that further development could lead to successful contact-active antialgal coatings.     

Enantioselective microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic acid, ethyl ester

The key chiral intermediate 3,5-dihydroxy-6-(benzyloxy) hexanoic acid, ethyl ester 2a, was made by the stereoselective microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic acid, ethyl ester 1. Among various microbial cultures evaluated, cell suspensions of Acinetobacter calcoaceticus SC 13876 reduced 1 to 2a. The reaction yield of 85% and optical purity of 97% was obtained using glycerol-grown cells. The substrate was used at 2 g l⁻¹ and cells were used at 20% (w/v, wet cells) concentrations. The optimum pH for the reduction of 1 to 2a was 5.5 and the optimum temperature was 32°C. Cell extracts of A. calcoaceticus SC 13876 in the presence of NAD⁺, glucose, and glucose dehydrogenase reduced 1 to the corresponding monohydroxy compounds 3 and 4 [3-hydroxy-5-oxo-6-(benzyloxy) hexanoic acid ethyl ester 3, and 5-hydroxy-3-oxo-6-(benzyloxy) hexanoic acid ethyl ester 4]. Both 3 and 4 were further reduced to 2a by cell extracts. Reaction yield of 92% and optical purity of 99% were obtained when the reaction was carried out in a 1-l batch using cell extracts. The substrate was used at 10 g l⁻¹. Product 2a was isolated from the reaction mixture in 72% overall yield. The GC and HPLC area % purity of the isolated product was 99% and the optical purity was 99.5%. The reductase which converted 1 to 2a was purified about 200-fold from cell extracts of A. calcoaceticus SC 13876. The purified enzyme gave a single protein band on SDS-PAGE corresponding to 35,000 daltons.     

Biological activities and structure-activity relationship of substitution compounds of N-[2-(3-indolyl)ethyl]succinamic acid and N-[2-(1-naphthyl)ethyl]succinamic acid, derived from a new category of root-promoting substances, N-(phenethyl)succinamic acid analogs

In a previous study, it was demonstrated that N-(phenethyl)succinamic acid (PESA) derivatives form a new category of root-promoting substances which do not exhibit auxin-like activities, such as stem elongation and leaf epinasty (Soejima et al., 2000 [Plant Cell Physiol. 41s: 197]). In this study, N-[2-(3-indolyl)ethyl]succinamic acid (IESA) and N-[2-(1-naphthyl)ethyl]succinamic acid (NESA) were synthesized, and their biological activities were evaluated. In an adzuki root-promoting assay, IESA and NESA exhibited root-promoting activity equivalent to PESA. In adzuki stem elongation assays, elongation activity was not observed in the stem segments soaked in either an IESA or NESA aqueous solution, whereas the stem segments immersed in Indole-3-acetic acid (IAA) or 1-naphthylacetic acid (NAA) aqueous solution were clearly elongated. In an epinastic bending study, IAA and NAA exhibited leaf epinasty, whereas IESA and NESA did not, suggesting that the IESA and NESA derivatives belong to the same category of root-promoting substances as PESA derivatives and are different from auxin-like substances. In addition, eleven kinds of IESA derivatives and nineteen kinds of NESA derivatives were synthesized, and their root-promoting activities were measured. The activities of methyl ester derivatives were approximately three times higher than that of the acid compounds, with exceptions for some compounds. The partition coefficient (P) between 1-octanol and water for each IESA, NESA, and PESA derivative was measured in order to evaluate the hydrophobicity of their molecules and to determine their structure–activity relationship. The results indicate that the root-promoting activity of the acid compounds was significantly correlated with their hydrophobicity, whereas that of ester derivatives was not correlated.     

High-performance liquid chromatographic determination of N-[2- (hydroxyethyl)-N-(2-(7-guaninyl)ethyl)]methylamine, a reaction product between nitrogen mustard and DNA and its application to biological samples

Nitrogen mustard (HN2) is a bifunctional alkylating agent which is thought to cause cytotoxicity by covalently binding to DNA. Most studies to date have looked at qualitatively determining the presence of DNA–HN2 adducts from reactions with native DNA. The adduct which is predominately formed in these reactions is N-[2-(hydroxyethyl)-N-(2-(7-guaninyl)ethyl]methylamine (N7G). A simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of N7G from DNA using ultraviolet detection is described. DNA samples having been exposed to HN2 treatment were hydrolyzed and preseparated from high-molecular-mass material by filtration using a molecular mass cut-off of 3000. The mobile phase consisted of methanol–26 mM ammonium formate, pH 6.5 (24:76, v/v). N7G, as well as the internal standard, methoxyphenol, were separated within 30 min. The recovery of N7G after hydrolysis of the DNA reaction product was quantitative and limits of detection and quantification of 10 and 20 ng/ml, respectively, were calculated. The method was validated in DNA–HN2 dose response experiments. The N7G reaction product appears to be the first reaction product formed at lower ratios of HN2/DNA but its production plateaus at higher ratios of HN2/DNA probably due to increased formation of hitherto unknown adducts. The method is simple and sensitive and for this reason, may be suited for the determination of DNA/HN2 reaction products.     

Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL)

BACKGROUND: Although some cationic reagents, such as polybrene, improve gene transduction in vitro, their use in vivo is prohibited due to their toxicity to the exposed cells. This paper demonstrates that a new cationic reagent, poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL), improves gene transduction with retroviral vectors without increasing cell toxicity. METHODS: A retroviral vector derived from the Moloney leukemia virus, containing the lacZ gene, was modified with PEG-PLL prior to transduction into NIH3T3, Lewis lung carcinoma, and primary cultured mouse brain cells. LacZ transduction efficacy was evaluated by counting the number of X-Gal-positive cells. RESULTS: We have demonstrated that PEG-PLL is able to stably modify the viral particle surface due to the affinity of the PEG moiety to the biomembrane, and neutralizes negative charges by the cationic nature of the poly-lysine residue. Thus, PEG-PLL increased the gene transduction efficiency and minimized cell toxicity because free PEG-PLL was removable by centrifugation. We have shown that PEG-PLL increased the viral gene transduction efficiency 3- to 7-fold with NIH3T3 or Lewis lung carcinoma cell lines without increasing cytotoxicity. It improved retroviral gene transduction efficacy even against labile cells, such as primary cultured brain cells. CONCLUSIONS: PEG-PLL is a novel reagent that improves retroviral gene transduction efficacy without increasing cytotoxicity.