采用BS-Ⅱ大孔离子交换树脂分离低分子肝素

采用HNO2降解法,通过控制降解过程中的pH和时间裂解低分子肝素,并经大孔离子交换树脂BS-Ⅱ对其进行分离纯化,用质谱法对其相对分子质量进行分析。结果表明:在pH3.5、4 h的降解条件下,降解得到低分子肝素。在0.34 mol/L NaCl、pH8.5的条件下吸附,4 mol/L NaCl、pH8.5条件下洗脱,其吸附率达97.2%,纯化后的低分子肝素抗凝活性为34.41 U/mg,比纯化前提高1.36倍,效价回收率达到85.24%。质谱分析表明,纯化所得低分子肝素的平均相对分子质量为8 411,67.45%集中分布在7 000~9 000段。     

Chitosan as an enabling excipient for drug delivery systems. I. Molecular modifications

Chitosan was physicochemically modified for its potential use as a matrix for an implantable antibiotic delivery system that could sustain bactericidal concentrations in the vicinity of an implant or prosthesis. Deacetylation and depolymerization of chitosan were implemented in order to increase the number or accessibility of the reactive amino groups on the polymer backbone for better polymer-drug interaction. The deacetylation process involved reaction of particulate chitosan/depolymerized chitosan with alkali. The rate of deacetylation of chitosan was directly proportional to the reaction temperature up to 80 degrees C; beyond 80 degrees C, rapid degradation of the polymer occurred. The depolymerization of chitosan involved acid digestion of the polymer followed by application of mechanical agitation. This depolymerized product, although water insoluble, possessed a molecular weight that was one to two orders of magnitude lower than that of commercially available chitosans. These products not only exhibited improved reactivity, but also showed increased crystallinity when compared with the parent chitosan. The reactivity was found to be inversely proportional to chitosan's molecular weight. The depolymerization and deacetylation treatments afforded formation of chitosan having a greater number of amino groups available for interactions with the anionic actives.     

Development and <Emphasis Type="Italic">In Vitro</Emphasis> Characterization of Galactosylated Low Molecular Weight Chitosan Nanoparticles Bearing Doxorubicin

The aim of the present research was to evaluate the potential of galactosylated low molecular weight chitosan (Gal-LMWC) nanoparticles bearing positively charged anticancer, doxorubicin (DOX) for hepatocyte targeting. The chitosan from crab shell was depolymerized, and the lactobionic acid was coupled with LMWC using carbodiimide chemistry. The depolymerized and galactosylated polymers were characterized. Two types of Gal-LMWC(s) with variable degree of substitution were employed to prepare the nanoparticles using ionotropic gelation with pentasodium tripolyphosphate anions. Factors affecting nanoparticles formation were discussed. The nanoparticles were characterized by transmission electron microscopy and photon correlation spectroscopy and found to be spherical in the size range 106–320 nm. Relatively higher percent DOX entrapment was obtained for Gal-LMWC(s) nanoparticles than for LMWC nanoparticles. A further increase in drug entrapment was found with nanoparticles prepared by Gal-LMWC with higher degree of substitution. A hypothesis which correlates the ionic concentration of DOX in nanoparticles preparation medium and percent DOX entrapment in cationic polymer has been proposed to explain the enhanced DOX entrapment. In-vitro drug release study demonstrated an initial burst release followed by a sustained release. The targeting potential of the prepared nanoparticles was assessed by in vitro cytotoxicity study using the human hepatocellular carcinoma cell line (HepG₂) expressing the ASGP receptors on their surfaces. The enthusiastic results showed the feasibility of Gal-LMWC(s) to entrap the cationic DOX and targeting potential of developed Gal-LMWC(s) nanoparticles to HepG₂ cell line.     

Kinetic study of acid depolymerization of chitosan and effects of low molecular weight chitosan on erythrocyte rouleaux formation

In this study, the depolymerization of chitosan was carried out in an acetic acid aqueous solution and was followed by viscometry for molecular weight determination. It was found that the depolymerization rate increased with elevated temperatures and with high acid concentrations. Based on FTIR analysis, the chitosan was depolymerized randomly along the backbone; no other structural change was observed during the acid depolymerization process. Revealed in the TGA study, the degradation temperature and char yield of LMWCs (low molecular weight chitosan) were molecular weight dependent. The blood compatibility of LMWCs was also investigated: rouleaux formation was observed when erythrocyte contacted with LMWCs, which showed that LMWCs are able to interfere with the negatively charged cell membrane through its polycationic properties. Furthermore, as regards a kinetics investigation, the values of M_n (number-average molecular weight) were obtained from an experimentally determined relationship. The kinetics study showed that the complex salt, formed by amine on chitosan and acetic acid, acted as catalyst. Finally, the activation energy for the hydrolysis of the glycosidic linkage on chitosan was calculated to be 40 kJ/mol; the mechanism of acid depolymerization is proposed. In summary, LMWCs could be easily and numerously generated with acid depolymerization for further biological applications.     

Low molecular weight derivatives of different carrageenan types and their antiviral activity

A number of low molecular weight (LMW) fractions of carrageenans with different structural types were obtained by free radical depolymerization (H₂O₂), mild acid hydrolysis (HCl), and a specific enzyme. Three samples of carrageenans were depolymerized: kappa-carrageenan from Chondrus armatus, kappa-carrageenan from Kappaphycus alvarezii, and kappa/beta-carrageenan from Tichocarpus crinitus with initial molecular weights of 250, 390, and 400 kDa, respectively. The chemical depolymerization by two methods resulted to LMW derivatives of carrageenans with molecular weight from 1.2 to 3.5 kDa. Oligosaccharides of kappa- and kappa/beta-carrageenans with molecular weight of 2.2 and 4.3 kDa, respectively, were obtained after enzymatic depolymerization by recombinant kappa-carrageenase from Pseudoalteromonas carrageenovora. It was shown that the antiviral activity of high molecular weight carrageenans against tobacco mosaic virus was higher than that of their LWM derivatives independently on the depolymerization method. The method of depolymerization had some influence on the antiviral activity of carrageenan. LMW derivatives of kappa- and kappa/beta-carrageenans obtained by mild acid hydrolysis showed higher antiviral activity than the products of free radical depolymerization. The oligosaccharides prepared by enzymatic degradation possessed the lowest activity.     

家养及大型养殖场鸡肠道微生物菌群及四环素耐药菌多样性的比较研究

本研究分析比较了农家家养鸡与大型养殖场鸡肠道菌群组成及抗四环素抗性基因在肠道菌群中的分布情况。通过454焦磷酸法对细菌16S rRNA V3区进行测序来分析肠道菌群的组成;用平板琼脂法筛选四环素耐药菌株,对其16S rRNA基因全长测序,并与RDP (Ribosomal Database Project)数据库比对进行菌种鉴定;聚合酶链反应(polymerase chain reaction,PCR)检测常见四环素耐药基因。家养鸡粪便中菌群香农多样性指数为5.321±0.590,养殖场鸡为4.398±0.440,前者显著大于后者(Mann-Whitney U test,P＝0.008)。从家养鸡和养殖场鸡粪便中随机分离到69株和65株四环素耐药菌株,后者四环素耐药菌的种类多于前者。家养鸡较养殖场鸡的肠道菌群更具多样性,而抗生素抗性基因在养殖场鸡的肠道菌群中分布更广泛。结果表明,不同饲养方式对鸡的肠道菌群有影响,对抗生素抗性基因的分布也有一定影响。     

Controlled Photochemical Depolymerization of K5 Heparosan, a Bioengineered Heparin Precursor

Heparosan is a polysaccharide, which serves as the critical precursor in heparin biosynthesis and chemoenzymatic synthesis of bioengineered heparin. Because the molecular weight of microbial heparosan is considerably larger than heparin, the controlled depolymerization of microbial heparosan is necessary prior to its conversion to bioengineered heparin. We have previously reported that other acidic polysaccharides could be partially depolymerized with maintenance of their internal structure using a titanium dioxide-catalyzed photochemical reaction. This photolytic process is characterized by the generation of reactive oxygen species that oxidize individual saccharide residues within the polysaccharide chain. Using a similar approach, a microbial heparosan from Escherichia coli K5 of molecular weight >15,000 was depolymerized to a heparosan of molecular weight 8,000. The (1)H-NMR spectra obtained showed that the photolyzed heparosan maintained the same structure as the starting heparosan. The polysaccharide chains of the photochemically depolymerized heparosan were also characterized by electrospray ionization-Fourier-transform mass spectrometry. While the chain of K5 heparosan starting material contained primarily an even number of saccharide residues, as a result of coliphage K5 lyase processing, both odd and even chain numbers were detected in the photochemically-depolymerized heparosan. These results suggest that the photochemical depolymerization of heparosan was a random process that can take place at either the glucuronic acid or the N-acetylglucosamine residue within the heparosan polysaccharide.     

The Effects of Hyaluronic Acid on Macrophage FC Receptor Binding and Phagocytosis Are Independent of the Mode of Depolymerization

In order to determine whether exposure of hyaluronic acid to oxygen radicals caused an alteration in its properties. independent of the change in molecular weight induced. we examined its effect upon macro-phage Fc receptor binding. High molecular weight hyaluronic acid (Healon-Pharmacia) caused a dose dependent inhibition of binding between the concentrations of 0.2–1 mg/ml. At a concentration of 0.3 mg/ml both oxygen radical depolymerized and enzymatically degraded hyaluronic acid caused an inhibition of Fc receptor binding at molecular weights of 1 × 10⁶. 1.5 × 10⁶ and 2 × 10⁶. Oxygen radical degraded hyaluronic acid caused a stimulation of Fc receptor binding at molecular weights of 2 × 10⁵ and 3.5 × 10⁵. and enzyme degraded hyaluronic acid causes stimulation at a molecular weight of 2.5 × 10⁶. Thus this “biological property” of hyaluronic acid is dependent upon molecular weight solely and not upon the mode of depolymerization.     

Characterization and blood coagulation evaluation of the water-soluble chitooligosaccharides prepared by a facile fractionation method

Water-soluble chitooligosaccharides have been reported to have specific biological activities. In this study, the chitosan samples with different degree of acetylation were used separately to prepare chitooligosaccharide (COS) and highly deacetylated chitooligosaccharide (HDCOS) through the nitrous acid depolymerization. Rather than using the conventional fractionation schemes commonly employed, such as dialysis and ultrafiltration which require a large amount of deionized water as well as a fair long dwell time, an unique fractionation scheme is explored to recover and desalt these nitrous-acid depolymerized chitosan with different molecular weights. This fractionation scheme is based on the differential solubility variation of depolymerized products within the aqueous solutions that contain various ratios of methanol. It was noted that chitosan with different molecular weight can be successfully recovered and fractionated with methanol added sequentially up to a volume of four times of original depolmerized product. In addition, chemical characterization of the fractionated water-soluble COS and HDCOS by 1H NMR spectroscopy and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) indicated that the chitosan depolymerization reaction is greatly influenced by the degree of acetylation of the parental chitosan reactant. Moreover, the modified whole blood clotting time assay and the platelet coagulation test suggested that the 1:2 fractionated water-soluble COS and HDCOS obtained are much less procoagulant than their parental chitosan compound and can be of use in biomedical applications in which blood coagulation is not desired.     

Peptide-based hydrogel nanoparticles as effective drug delivery agents

Peptide-based hydrogel nanoparticles represent a promising alternative to current drug delivery approaches. We have previously demonstrated that the Fmoc-FF aromatic dipeptide building block can self-assemble in aqueous solutions to form nano-scaled ordered hydrogels of remarkable mechanical rigidity. Here, we present a scalable process for the assembly of this peptide into hydrogel nanoparticles (HNPs) aimed to be utilized as potential drug delivery carriers. Fmoc-FF based HNPs were formulated via modified inverse-emulsion method using vitamin E-TPGS as an emulsion stabilizer and high speed homogenization. The formed HNPs exhibited two distinguishable populations with an average size of 21.5 ± 1.3 and 225.9 ± 0.8 nm. Gold nanoparticles were encapsulated within the hydrogel nanoparticles as contrast agents to monitor the formation of the assemblies and their ultrastructural properties. Next, we demonstrated a robust experimental procedure developed and optimized for the formulation, purification, storage and handling procedures of HNPs. Encapsulation of doxorubicin (Dox) and 5-flourouracil (5-Fu) within the HNPs matrix showed release kinetics of the drugs depending on their chemical structure, molecular weight and hydrophobicity. The results clearly indicate that Fmoc-FF based hydrogel nanoparticles have the potential to be used as encapsulation and delivery system of various drugs and bioactive molecules.     

Partial depolymerization of pectin by a photochemical reaction

Complex heterogeneous polysaccharides that comprise pectin were partially depolymerized by a photochemical reaction using ultraviolet light in the presence of titanium dioxide catalyst. In a period of 6 h at pH 7, this UV/TiO₂ process decreased the average molecular weight of pectin from 400 kDa to 200 kDa. The characterization of the partially depolymerized pectin, which was fractionated by size-exclusion chromatography, was performed by ¹H NMR spectroscopy, and the spectra obtained showed that the resulting oligosaccharides and polysaccharides maintained the intact core structure of pectin. The monosaccharide content and depolymerization profile were determined by high-performance anion-exchange chromatography coupled with pulsed amperometric detection. This controlled photochemical depolymerization technique might be useful for preparation of pectin oligosaccharides as an ingredient in food and pharmaceutical products.     

Intracellular Delivery of Nanoparticles of an Antiasthmatic Drug

The aim of the investigation was to prepare and characterize wheat germ agglutinin(WGA)-conjugated poly(d,l-lactic-co-glycolic) acid nanoparticles encapsulating mometasone furoate (MF) as a model drug and assess changes in its fate in terms of cellular interactions. MF loaded nanoparticles were prepared using emulsion–solvent evaporation technique. WGA-conjugation was done by carbodiimide coupling method. The nanoparticles were characterized for size, zeta potential, entrapment efficiency and in-vitro drug release. The intracellular uptake of nanoparticles, drug cellular levels, and anti-proliferative activity studies of wheat germ agglutinin-conjugated and unconjugated nanoparticles were assessed on alveolar epithelial (A549) cells to establish cellular interactions. Prepared nanoparticles were spherical with 10–15 μg/mg of WGA conjugated on nanoparticles. The size of nanoparticles increased after conjugation and drug entrapment and zeta potential reduced from 78 ± 5.5% to 60 ± 2.5% and −15.3 ± 1.9 to −2.59 ± 2.1 mV respectively after conjugation. From the cellular drug concentration–time plot, AUC was found to be 0.4745, 0.6791 and 1.24 for MF, MF-nanoparticles and wheat germ agglutinin-MF-nanoparticles respectively. The in-vitro antiproliferative activity was improved and prolonged significantly after wheat germ agglutinin-conjugation. The results conclusively demonstrate improved availability and efficacy of antiasthmatic drug in alveolar epithelial cell lines. Hence, a drug once formulated as mucoadhesive nanoparticles and incorporated in dry powder inhaler formulation may be used for targeting any segment of lungs for more improved therapeutic response in other lung disorders as well.     

Photolytic depolymerization of alginate

A photochemical reaction has been developed for the partial de-polymerization of sodium alginate, a polysaccharide utilized in medicine, pharmacy, basic sciences and foods. An aqueous solution of sodium alginate was photochemically depolymerized to ∼40% of its average molecular weight using ultraviolet light in the presence of titanium dioxide catalyst at pH 7 over a period of 3 h. The products were separated giving four fractions all having an average molecular weight that was smaller than that of the starting material. Characterization of the guluronate (G) and mannuronate (M) contents, and determination of the M/G ratio of photochemically depolymerized alginate, were accomplished using ¹H NMR spectroscopy. The resulting M/G ratio was compared to that obtained for alginate fractions produced by acid hydrolysis. The M and G content, of each alginate fraction, was also assigned with regards to their occurrence in G-rich, M-rich or M/G heteropolymeric domains. This new depolymerization method might also be applicable in the preparation of alginate oligosaccharides for use in the food and pharmaceutical industries.     

长春新碱阳离子纳米结构脂质载体及其小肠吸收的研究

目的:硫酸长春新碱作为一种细胞毒型抗肿瘤药物,临床上多用其注射剂,虽应用广泛,但存在较多缺点,如药物半衰期短,代谢速率快以及毒副作用明显。本文目的是制备包载长春新碱和十二烷基磺酸钠的阳离子纳米结构脂质载体,并对其进行评价。方法:用复乳挥发法制备出目标脂质纳米粒;利用激光粒度仪对其粒径及zeta电位进行检测;利用高效液相色谱法对其包封率和载药量进行测定;透析法检测纳米粒的体外释放行为;用小肠吸收法评价纳米粒的促进吸收作用。结果:制得的纳米粒的平均粒径为(192.4±4.14)nm,多分散系数(PDI)为0.184±0.015,包封率为32.28%,Zeta电位为(30.6±4.09)m V,载药量为(1.56±0.10)%;体外释放实验显示在pH=7.4的中性释放介质中,硫酸长春新碱脂质纳米粒表现出缓释特性;小肠吸收实验表明十二烷基磺酸钠的加入和阳离子纳米粒的修饰可提高小肠对药物的吸收。结论:阳离子硫酸长春新碱纳米结构脂质载体具有缓释效果,并可以促进小肠对药物的吸收。     

Depolymerization of glycosaminoglycuronans into di- and higher molecular-weight oligo-saccharides: Improved preparation of N-acetyldermosine and oligomeric N-acetylchondrosines

Nonsulfated di- to octadeca-saccharides having 2-acetamido-2-deoxy-d-galactose at the reducing end were prepared, in 81% yield, by treatment of chondroitin 6-sulfate (pyridinium salt) with dimethyl sulfoxide containing 10% of water for 14 h at 90°. N-Acetylchondrosine and N-acetyldermosine were obtained from dermatan sulfate of rooster comb, in 30% and 38% yields, respectively, by solvolysis with dimethyl sulfoxide, containing 10% of water, for 30 h at 105°. Hyaluronic acid was also depolymerized by the same solvent in the presence of an equimolar amount of pyridinium sulfate or chloride per disaccharide unit to give reducing di- and higher molecular weight oligo-saccharides. The results of solvolytic desulfation and depolymerization are compared with those of the conventional methods by acid hydrolysis.     

Ozonolytic depolymerization of polysaccharides in aqueous solution.

The selective oxidation of beta-D-glycosidic linkages of polysaccharides by ozone has great utility as a general method for depolymerization of polysaccharides. Here we describe a 'one-step' method whereby polysaccharides dissolved in water or basic solutions are depolymerized by ozonolysis. The oxidation of glycosidic linkages of unprotected carbohydrates by ozone is complicated by several side reactions. We describe here optimized conditions for carrying out ozonolysis degradation. We also characterize the major pathways for unwanted degradation by various side reactions. In the preferred oxidation pathway, the aldosidic linkage is oxidized to an aldonic ester function that hydrolyzes under the basic conditions employed to give a free aldonate, with cleavage of the polysaccharide chain. Nonselective degradation pathways include oxidative degradation by radical species that oxidize glycosyl residues to formic, acetic, and oxalic acids. The nonselective degradation caused by acids is minimized by basic buffers. The products of polysaccharide depolymerization form a size distribution around a nominal molecular weight, and the average molecular weight of the products can be controlled by the rate or amount of ozone passed through the reaction mixture. The ozonolysis method described herein provides a convenient, inexpensive, and controllable means for generating small polysaccharides or large oligosaccharide fragments.     

Molecular dynamics simulation of drug uptake by polymer

Drug uptake by polymer was modeled using a molecular dynamics (MD) simulation technique. Three drugs—doxorubicin (water soluble), silymarin (sparingly water soluble) and gliclazide (water insoluble)—and six polymers with varied functional groups—alginic acid, sodium alginate, chitosan, Gantrez AN119 (methyl-vinyl–ether-co-malic acid based), Eudragit L100 and Eudragit RSPO (both acrylic acid based)—were selected for the study. The structures were modeled and minimized using molecular mechanics force field (MM+). MD simulation (Gromacs-forcefield, 300 ps, 300 K) of the drug in the vicinity of the polymer molecule in the presence of water molecules was performed, and the interaction energy (IE) between them was calculated. This energy was evaluated with respect to electric-dipole, van der Waals and hydrogen bond forces. A good linear correlation was observed between IE and our own previous data on drug uptake^* [R ² = 0.65, R_adj² = 0.65,R_pre² = 0.56, {\hbox{R}}_{\rm{adj}}^2 = 0.65,{\hbox{R}}_{\rm{pre}}^2 = 0.56, and a F ratio of 30.25, P < 0.001; Devarajan et al. (2005) J Biomed Nanotechnol 1:1–9]. Maximum drug uptake by the polymeric nanoparticles (NP) was achieved in water as the solvent environment. Hydrophilic interaction between NP and water was inversely correlated with drug uptake. The MD simulation method provides a reasonable approximation of drug uptake that will be useful in developing polymer-based drug delivery systems.     

用于核酸递送的GSH响应型纳米粒构建

摘要 目的：核酸治疗近年来越来越受到关注,但是核酸药物易被快速清除、易被核酸酶降解、非特异性生物分布、以及不易被细胞摄取的缺点使其在体内难以发挥效果。本文提供了一种具有谷胱甘肽（GSH）响应性释放的纳米粒,能够进行有效核酸药物递送。方法：使用十六烷基三甲基氯化铵（CTAC）制备介孔硅纳米粒,在介孔硅纳米粒表面进行巯基修饰并活化,使其与巯基修饰的聚丙烯亚胺和聚乙二醇反应,形成具有GSH响应的介孔硅纳米粒,通过静电吸附进行核酸荷载。马尔文粒度仪测量表面电位、粒径,透射电镜观察纳米粒形态。核酸电泳检测其核酸负载效率,通过体外检测GSH响应释放聚乙烯亚胺（PEI）情况,共聚焦显微镜观察细胞摄取以及溶酶体逃逸情况。结果：成功构建了具有GSH响应的纳米粒,粒径为76.44±1.68 nm,表面电位为33.93±0.59 mV;通过透射电镜观察到纳米粒呈圆形带孔颗粒状;琼脂糖核酸负载试验观察到当氮磷比大于20时,能够有效进行核酸负载。共聚焦显微镜显示该纳米粒能够成功被MDA-MB-231乳腺癌细胞摄取。在溶酶体逃逸试验中观察到纳米粒进入细胞后3 h,Cy5-siRNA与溶酶体的荧光分离,证明构建的纳米粒成功从溶酶体逃脱。结论：成功构建了具有GSH响应的介孔硅纳米粒,能够有效用于核酸递送。     

Optimization of methazolamide-loaded solid lipid nanoparticles for ophthalmic delivery using Box–Behnken design

The purpose of the present study was to optimize methazolamide (MTZ)-loaded solid lipid nanoparticles (SLNs) which were used as topical eye drops by evaluating the relationship between design factors and experimental data. A three factor, three-level Box–Behnken design (BBD) was used for the optimization procedure, choosing the amount of GMS, the amount of phospholipid, the concentration of surfactant as the independent variables. The chosen dependent variables were entrapment efficiency, dosage loading, and particle size. The generated polynomial equations and response surface plots were used to relate the dependent and independent variables. The optimal nanoparticles were formulated with 100?mg GMS, 150?mg phospholipid, and 1% Tween80 and PEG 400 (1:1, w/v). A new formulation was prepared according to these levels. The observed responses were close to the predicted values of the optimized formulation. The particle size was 197.8?±?4.9?nm. The polydispersity index of particle size was 0.239?±?0.01 and the zeta potential was 32.7?±?2.6?mV. The entrapment efficiency and dosage loading were about 68.39% and 2.49%, respectively. Fourier transform infrared spectroscopy (FT-IR) study indicated that the drug was entrapped in nanoparticles. The optimized formulation showed a sustained release followed the Peppas model. MTZ-SLNs showed significant prolonged decreasing intraocular pressure effect comparing with MTZ solution in vivo pharmacodynamics studies. The results of acute eye irritation study indicated that MTZ-SLNs and AZOPT both had no eye irritation. Furthermore, the MTZ-SLNs were suitable to be stored at low temperature (4?°C).     

甘油三酯介质中制备壳聚糖纳米粒工艺研究

用乳化溶剂扩散法结合离子沉淀交联法从甘油三酯介质中制备壳聚糖纳米粒,用L9(34)正交设计优选纳米粒制备的处方工艺条件,用显微镜测定纳米粒的粒径,用透射电镜观察纳米粒的形态。结果:正交设计确定纳米粒制备的最优处方工艺条件为:搅拌速度150 r.min-1,壳聚糖质量分数0.10%,壳聚糖分子量9.1万,甘油三酯与壳聚糖酸溶液体积之比200:1,制备的纳米粒平均粒径为(150±50)nm。甘油三酯介质中制备壳聚糖纳米粒工艺简便,制剂具有广泛应用前景。