PEG化壳聚糖/DNA自组装复合物的制备、表征和体外Hela细胞转染研究

通过有机合成和高分子聚合等方法将亲水性的聚乙二醇接枝到壳聚糖的氨基侧链上，得到了改性的壳聚糖—聚乙二醇接枝共聚物，应用现代波谱等技术对中间产物和最终产物进行了表征，采用绿色荧光蛋白基因质粒pEGFP—N1为DNA模型，在溶液中通过自动(静电)吸附得到PEG化的壳聚糖／DNA自组装复合物，初步研究了该自组装复合物对Hela细胞的体外转染效率。结果表明，活化的聚乙二醇被成功地接枝到壳聚糖上，使不溶于水的壳聚糖改性为水溶性的PEG化的壳聚糖。PEG化壳聚糖／DNA自组装复合物在Hela细胞体外转染率达到81％。因此，PEG化的壳聚糖有可能成为基因转染的非病毒载体。     

聚乙二醇改性壳聚糖作为小分子药物载体的研究进展

壳聚糖具有抗菌、抗氧化、增强胶凝特性以及可作为生物活性分子的微型或纳米载体等优点,因此其化学改性和应用近年来受到广泛关注。然而,壳聚糖既不溶于有机溶剂也不溶于水,极大地限制了它的应用。在改性的壳聚糖中,聚乙二醇化壳聚糖不仅能保持壳聚糖的优点,还能提高水溶性,并能有效运输生物活性分子。因此,本文总结了2008-2012年聚乙二醇化壳聚糖作为紫杉醇、阿霉素、5-氟尿嘧啶等小分子载体的最新进展,为今后聚乙二醇化壳聚糖的研究提供有益参考和理论依据。     

聚乙二醇改性壳聚糖作为小分子药物载体的研究进展

壳聚糖具有抗菌、抗氧化、增强胶凝特性以及可作为生物活性分子的微型或纳米载体等优点,其化学改性和应用近年来受到广泛关注。然而,壳聚糖既不溶于有机溶剂也不溶于水,极大地限制了它的应用。在改性的壳聚糖中,聚乙二醇化壳聚糖不仅能保持壳聚糖的优点,还能提高水溶性,并能有效运输生物活性分子。本文综述了近5年间聚乙二醇化壳聚糖作为紫杉醇、阿霉素、5-氟尿嘧啶等小分子载体的研究进展,为今后的研究提供有益参考和理论依据。     

mCMC-PEG/mCS-PEG双极膜的制备与表征

以Fe3+改性羧甲基纤维素(mCMC)和聚乙二醇(PEG)共混为阳膜;以戊二醛改性壳聚糖(mCS)和聚乙二醇共混为阴膜,制备了mCMC-PEG/mCS-PEG双极膜.以FTIR测定了膜红外光谱,以扫描电镜观察了膜表面和界面层的形态,以TG进行膜的热重分析.测定了mCMC-PEG和mCS-PEG不同比例共混膜的含水率、离子交换容量、溶胀度,及mCMC-PEG/mCS-PEG双极膜的电性能.研究结果表明,在双极膜材料中引入亲水性的聚乙二醇后,因分子间的相容性增大,故而提高了双极膜的离子交换容量,并减小了膜的溶胀性.当CMC∶PEG质量比等于10∶1和CS∶PEG质量比等于2∶1时所制得的双极膜具有良好的电化学性能,在酸碱溶液中机械强度高、溶胀小.     

金纳米粒子/壳聚糖/石墨烯纳米复合材料的制备及其生物相溶性

利用壳聚糖(CTS)的氨基对石墨烯(GNS)进行表面改性制得石墨烯基纳米复合材料(E).利用E中CTS对金纳米粒子(Au)良好的保护作用,在E的表面固载Au制备了Au/CTS/GNS纳米复合材料(F),其结构经UV-Vis和FT-IR表征.并用Zeta电位,XRD和TEM对F的性质进行了研究.结果表明,F不仅具有良好的生物相溶性,而且具有较好的导电性能.     

聚乙二醇型超支化聚酯胺的制备及表征

以M_n=250的聚乙二醇二丙烯酸酯为A_2单体(含有两个A基因的单体,其余类推),N-乙基乙二胺为B'B_2单体,利用丙烯酸酯基和胺基的迈克尔加成聚合反应,一锅法合成了表面含丙烯酸酯键、骨架含聚乙二醇(PEG)、内部含叔胺的具有三维椭球状结构的超支化聚酯胺,并研究了其聚合动力学。利用咪唑对超支化聚酯胺进行改性,并初步研究了咪唑改性的超支化聚酯胺的pH响应性和水溶性。利用核磁共振氢谱、红外光谱、凝胶渗透色谱、GPC-MALLS多角度激光散射系统表征了超支化聚酯胺的分子结构和化学组成。结果表明:聚乙二醇链段的引入能够赋予超支化聚酯胺水溶性;咪唑的引入有利于提高超支化聚酯胺的pH响应性。     

壳聚糖改性的高分子表面活性剂的合成与性能

以商品壳聚糖(CTS)为原料,通过对-NH_2的化学改性,合成制备了壳聚糖改性的水溶性两性离子表面活性剂.采用正交试验法对合成反应工艺作了优化;通过红外光谱(IR)、核磁共振(H-NMR)及元素分析(EA)等方法对烷基壳聚糖中间体(RCTS)与最终产物,即烷基壳聚糖磺酸季铵盐(SRCTS)的分子结构进行了表征,并对SRCTS的水溶性、表面张力以及泡沫性能作了初步的探索.结果表明:经改性的SRCTS具有良好的表面活性与泡沫稳定性,其最低表面张力值可降至29.1mN/m;泡沫半衰期可提高65%.     

庚醛改性壳聚糖的制备及其对酚类化合物的吸附性能

在相转移催化剂存在下由庚醛与壳聚糖反应生成Schiff's碱,再用NaBH4 还原制备了N-烷基化壳聚糖衍生物,改性壳聚糖(CTS)产物的结构用FTIR和XRD进行了表征,研究了它对2,4-二氯酚的吸附性能. 考察了吸附时间、溶液pH值、2,4-二氯酚浓度和改性剂用量等因素对吸附的影响. 结果表明,改性CTS具有较好的抗酸碱性能;溶液的pH值对吸附的影响较大,在pH=6.0,吸附2 h时对2,4-二氯酚的吸附量最大,酚浓度对吸附的影响符合Freundlich吸附等温方程;改性壳聚糖对2,4-二氯酚的吸附性能明显优于未改性的CTS,对质量浓度为0.6 g/L的2,4-二氯酚溶液的吸附量分别为70.0和7.7 mg/g.     

壳聚糖与聚乙二醇交联水凝胶研究

研究对壳聚糖(CS)进行化学修饰得到了不同丙烯酰基取代度(1.03%,3.55%和5.21%)的丙烯酰化羟丙基壳聚糖(AHCS);通过自由基引发反应,AHCS与聚乙二醇二丙烯酸酯(PEGDA)交联得到壳聚糖(CS)与聚乙二醇(PEG)为主体的交联水凝胶。通过SEM观察其为通透性良好的多孔性支架材料。水凝胶的溶胶含量和溶胀度随丙烯酰基取代度的增加而降低,水凝胶中壳聚糖的降解速率也随丙烯酰基取代度的升高而降低。对于同一取代度的交联水凝胶,其在酸性和碱性条件下的溶胀度大于中性环境。细胞试验表明,壳聚糖与聚乙二醇交联水凝胶具有良好的生物相容性。     

单一分布聚乙二醇的合成及其应用

聚乙二醇(PEG)经过对端基官能团化接枝到药物等生物活性分子上后,可提高药物等的水溶性、抗免疫原性、生物相容性和稳定性并降低毒性。工业级聚乙二醇由于杂质含量高尤其是其中二醇的含量过高、分子量分布过宽,不能直接用于药物等的改性。除利用聚合方法制备窄分布高纯度的聚乙二醇外,采用有机合成方法合成单一聚乙二醇链是有效的手段。对"单一链长(discrete length)"或者"单一分布(mono-disperse)"的聚乙二醇高分子聚合物的近年来主要合成方法的进展进行了综述并简介了其主要应用。     

Preparation of the water-soluble chitosan-coated oxidized regenerated cellulose gauze

A water-soluble chitosan-coated oxidized regenerated cellulose (ORC) gauze was prepared by the oxidation of a viscose gauze with NO₂/CCl₄ and subsequent treatment with a solution of chitosan in aqueous acetic acid and finally neutralization with NaOH/C₂H₅OH solution. A series of C6 ORC samples with different –COOH content were prepared and coated by chitosans (CTS) with different molecular weight (Mw) of 2,000, 50,000, 100,000 (denoted as CTS1, CTS2, CTS3). FT-IR and TG suggested the formation of the amide bond between the carboxyl group of ORC and the amino group of CTS. Kjeldahl nitrogen analysis of ORC gauze treated with CTS (CTS-ORC) showed that the percentage of chitosan with the lowest Mw of 2,000 introduced on ORC surface was highest and increased with oxidation time, while chitosans with medium and high Mw showed that the maximum percentage of chitosan introduced on ORC surface occurred at the oxidation time of 8 h. The neutralized chitosan-coated ORC gauze could still maintain its original morphological form and was water-soluble, and could form a transparent gel quickly for 5 s in water. The prepared water-soluble gauze could be anticipated to possess the improved hemostatic and antibacterial properties.     

Supramolecular hydrogel formation based on inclusion complexation between poly(ethylene glycol)-modified chitosan and alpha-cyclodextrin

Supramolecular hydrogels have been prepared on the basis of polymer inclusion complex (PIC) formation between poly(ethylene glycol) (PEG)-modified chitosans and alpha-cyclodextrin (alpha-CD). A series of PEG-modified chitosans were synthesized by coupling reactions between chitosan and monocarboxylated PEG using water-soluble carbodiimide (EDC) as coupling agent. With simple mixing, the resultant supramolecular assembly of the polymers and alpha-CD molecules led to hydrogel formation in aqueous media. The supramolecular structure of the PIC hydrogels was confirmed by differential scanning calorimetry (DSC), X-ray diffraction, and (13)C cross-polarized/magic-angle spinning (CP/MAS) NMR characterization. The PEG side-chains on the chitosan backbones were found to form inclusion complexes (ICs) with alpha-CD molecules, resulting in the formation of channel-type crystalline micro-domains. The IC domains play an important role in holding together hydrated chitosan chains as physical junctions. The gelation property was affected by several factors including the PEG content in the polymers, the solution concentration, the mixing ratio of host and guest molecules, temperature, pH, etc. All the hydrogels in acidic conditions exhibited thermo-reversible gel-sol transitions under appropriate conditions of mixing ratio and PEG content in the mixing process. The transitions were induced by supramolecular association and dissociation. These supramolecular hydrogels were found to have phase-separated structures that consist of hydrophobic crystalline PIC domains, which were formed by the host-guest interaction between alpha-CD and PEG, and hydrated chitosan matrices below the pK(a).The formation of inclusion complexes between alpha-cyclodextrin and PEG-modified chitosan leads to the formation of hydrogels that can undergo thermo-reversible supramolecular dissociation.     

光纤折射率传感用于壳聚糖脱乙酰度测定

建立了一种基于光纤折射率传感技术的壳聚糖脱乙酰度测定方法. 利用光纤折射率传感器监测酸碱滴定过程中溶液折射率的变化, 根据折射率变化转折点之间碱的用量来计算壳聚糖的脱乙酰度. 该方法测得的3种不同含量实际样品的脱乙酰度与氢核磁共振波谱(¹H NMR)方法测定结果相符, 验证了方法的可靠性. 该方法具有用量少、 结构简单、 准确、 重复性好和转折点明显等优点, 可应用于工业生产中壳聚糖脱乙酰度的测定.     

The formation of conjugates with PEG–chitosan improves the biocatalytic efficiency and antitumor activity of L-asparaginase from <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

The ChitoPEGylation method, which is a novel approach to regulating the catalytic properties of enzymes that is based on the formation of a covalent conjugate of an enzyme with branched copolymers of chitosan, has been developed. The efficiency of this method has been demonstrated using a new recombinant preparation of L-asparaginase from Erwinia carotovora (EwA) as a model. The molecular architecture and composition of EwA conjugates with PEG–chitosans have been optimized. It has been shown that the decisive factors that affect the activity of the EwA conjugates are the molecular weight of and PEGylation degree of chitosan. It has been found that the EwA conjugation with PEG–chitosan increases, its cytostatic activity against human chronic myeloid leukemia K562 cells, Burkitt’s lymphoma Raji cells, and acute lymphoblastic leukemia Jurkat cells. These data provide new approaches to the synthesis of L-asparaginase preparations with improved biocatalytic properties.     

壳多糖抑制细菌生长的构效关系

运用化学结构已清楚, 分属4大系列的29种壳多糖, 以4种不同类型的细菌(革兰氏阳性菌Ecoli K1、革兰氏阴性菌Bacillus cereus、Bacillus megaterium和Staphlylococcu aureus)为研究对象, 进行了壳多糖抑菌能力构效关系的研究. 在实验中采用96孔平板, 用计算机\|吸光值读数仪直接测定每个孔的吸光值, 获得了各个细菌在不同壳多糖浓度中的生长曲线和壳多糖抑制细菌生长的最低抑制浓度(MIC, Minimum inhibit concentration). 通过比较同一(各个)系列的壳多糖在这些相同(不同)细菌的MIC变化规律与壳多糖的化学结构的关系, 发现同一壳多糖对不同的细菌的MIC值是不相同的, 因而壳多糖抑制细菌生长的能力首先与细菌本身特点有关, 但与是否为革兰氏阳性菌或阴性菌无直接的相关性; 同一细菌对不同化学结构的壳多糖有一定的相关性, 在壳多糖的聚合程度(DP)相同的条件下, 壳多糖中氨基被乙酰化(DA)的程度越低, 壳多糖抑制细菌生长的MIC值越低, 壳多糖抑制细菌生长的能力就越强; 同样,在DA相同的情况下, 分子越小, 壳多糖抑制细菌生长的MIC值越低, 抑制细菌生长的能力越强. 根据上述实验结果, 初步推测壳多糖抑制细菌生长的机制可能与其在溶液中所带的正电荷多少有关.     

羧甲基壳聚糖微球对活性艳红X-3B的吸附行为研究

用交联的壳聚糖微球(CTS)与氯乙酸在碱性条件下反应,合成了羧甲基壳聚糖树脂(CMCT)。其吸附染料活性艳红X-3B的实验结果表明,CMCT和CTS均对偶氮染料活性艳红X-3B有较好的去除能力。实验条件下,最大平衡吸附量分别为611.5mg/g和365.2mg/g,说明羧甲基的引入提高了壳聚糖的吸附能力。等温吸附可以用Langmuir方程较好的描述,表明为单分子层吸附。动力学过程用二级吸附动力学模拟具有很好的线性相关性,通过二级吸附模型计算出的平衡吸附量与实验值相符。流动床实验表明,CMCT和CTS对浓度为100mg/L的X-3B溶液吸附的穿透点分别为6000ml/g和3375ml/g,用0.1mol/L的氢氧化钠溶液洗脱,洗脱峰集中,洗脱率都在90%以上。洗脱再生后的CMCT和CTS树脂均可重复使用。     

茜素红S与壳聚糖相互作用的电化学行为研究

在pH5.0NaAc—HAc缓冲溶液中,茜素红S能与壳聚糖生成一种紫红色的复合物。复合物使茜素红S在-0.408 V处的还原峰峰电流降低。在选定的最佳条件下,用循环伏安法和线性扫描极谱法测定其峰电流的降低值与壳聚糖的浓度在0.5～7．5mg/L的范围内呈线性关系,线性回归方程为△ip^#=30．92 108．4CCTS(mg／L),相关系数0.9990;检出限0．4mg/L。据此建立一种快速、简便测定壳聚糖的方法。该方法可应用于实际样品的测定。     

Synthesis of chitosan derivatives supported palladium complexes and their catalytic behavior in the Heck reaction

Two kinds of chitosan derivatives, crosslinked chitosan and crosslinked chitosan condense with salilylaldehyde, supported palladium complexes (CL‐CTS‐Pd and CL‐S‐CTS‐Pd) were synthesized and characterized by X‐ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA), differential thermal analysis (DTA), etc. These complexes are efficient catalysts for the Heck reaction under atmospheric conditions and can be easily recovered and reused. The detailed studies show that the catalyst CL‐S‐CTS‐Pd is much more efficient than CL‐CTS‐Pd under the same conditions. CL‐S‐CTS‐Pd keeps its catalytic activity in the Heck reaction of acrylic acid with iodobenzene even at a low temperature (60°C) or with tiny amounts of the catalyst (0.05 mol%Pd). Yields of making cinnamic acid were even as high as 75.3% in the Heck reaction of acrylic acid with iodobenzene using CL‐S‐CTS‐Pd that was recovered 10 times. Copyright © 2005 John Wiley & Sons, Ltd.     

Altered enzymatic activity of lysozymes bound to variously sulfated chitosans

The purpose of this research is to investigate the effects of the variously sulfated chitosans on lysozyme activity and structure.It was shown that the specific enzymatic activity of lysozyme remained almost similar to the native protein after being bound to 6-O-sulfated chitosan(6S-chitosan) and 3,6-O-sulfated chitosan(3,6S-chitosan),but decreased greatly after being bound to 2-N-6-O-sulfated chitosan(2,6S-chitosan).Meanwhile,among these sulfated chitosans,2,6S-chitosan induced the greatest conformational change in lysozyme as indicated by the fluorescence spectra.These findings demonstrated that when sulfated chitosans of different structures bind to lysozyme,lysozyme undergoes conformational change of different magnitudes,which results in corresponding levels of lysozyme activity.Further study on the interaction of sulfated chitosans with lysozyme by surface plasmon resonance(SPR) suggested that their affinities might be determined by their molecular structures.     

Elucidation of PEGylation site with a combined approach of in-source fragmentation and CID MS/MS

Poly(ethylene glycol) (PEG)ylation of peptides and proteins creates significant challenges for detailed structural characterization, such as PEG heterogeneity, site of addition and number of attached PEGylated moieties. Recently, we published a novel LC/MS methodology with a post-column addition of amines to obtain accurate masses of PEGylated peptides and proteins. The accurate masses can be used to assign the structures and number of attached PEGs [15], but the PEGylation site remains unclear in situations where multiple potential attachments are involved. Here, we present a methodology combining in-source fragmentation (ISF) with CID-MS/MS to elucidate the PEGylated sites in PEGylated products. All PEGylated samples, either prepared in acidic solution, or collected from a RP-HPLC stream, were first ionized via ISF to produce products containing small PEG fragment attachment, and then those fragment ions obtained were sequenced via CID MS/MS to deduce the PEGylation site. The methodology was successfully applied to PEGylated glucagon and IgG4 antibody light chain, which demonstrated that the small PEG fragments attached were stable during the CID activation.