聚乙二醇共价修饰药用蛋白质的分析方法

聚乙二醇共价修饰蛋白质具有重要的生物学应用意义。分析聚乙二醇共价修饰蛋白质对确定产物的修饰程度是必要的,近年来渐成研究热点。主要介绍了聚乙二醇共价修饰蛋白质的各种分析方法与其优缺点。同时对不同原理的分析方法进行了分类比较。     

聚合物蛋白质类药物单分子修饰的发展和展望

随着基因组学、生物化学、分子生物学及多肽合成等科学技术的发展,蛋白质及多肽药物已成为跨国医药企业竞相争夺未来生物医药领域的一个制高点。但是蛋白质类药物的临床应用受到了其理化及生物特性的种种限制,为了改善治疗效果,近年来聚合物修饰的蛋白质类药物有了长足的发展。本文着重回顾了蛋白质类药物聚乙二醇修饰的发展和现状,以及由此获得的优点和局限性;同时介绍了其它新型生物相容性聚合物蛋白质类药物修饰的最新研究进展,探讨了蛋白质类药物的"后聚乙二醇修饰"时代的可能方向和应用前景。     

树形大分子基药物传输系统结合强度及构型的分子动力学研究

采用全原子分子动力学方法系统研究了聚酰胺(PAMAM)型树形大分子非共价搭载4种抗癌药物分子(CE6,DOX,MTX及SN38)的药物传输复合体系.考察了药物分子种类、数量及树形大分子的代数和聚乙二醇化表面修饰对复合体系的结合强度、尺寸及溶剂中扩散行为的影响.研究发现,PAMAM自身变形能对药物-PAMAM间的结合有重要影响.搭载较多的药物分子可以使PAMAM自身增大,但同样搭载条件下经过聚乙二醇化修饰过的PAMAM变化并不明显.PAMAM分子表面的聚乙二醇化可以更高的强度结合更多的药物分子,并减缓其扩散速度,因而提高药物分子的搭载效率和体内滞留时间.为新型树形大分子基药物传输体系的设计提供理论依据.     

多肽和蛋白质的聚乙二醇化修饰方法

聚乙二醇是一类具有独特理化性质的大分子聚合物。多肽和蛋白质类药物经聚 乙二醇共价修饰后能明显改善其药代学和药效学性质，如降低免疫原性、增加对蛋 白水解酶的稳定性、增加水溶性及延长体内的半衰期等。蛋白质的聚乙二醇化修饰 研究已取得较好的效果，多肽的聚乙二醇化修饰研究起步较晚。对近年来多肽和蛋 白质的聚乙二醇化修饰方法进行了综述，主要介绍了对多肽和蛋白质的N端、C端及 某些氨基酸侧链进行选择性聚乙二醇化修饰的方法。     

聚乙二醇在新型药物制剂中的应用

聚乙二醇具有良好的生物相容性和两亲性 ,在生物医药领域中有着广泛的应用 ,本文就聚乙二醇在新型药物制剂中的应用进行综述 ,主要包括纳米给药系统、蛋白质药物修饰和疏水性药物的前药等。     

蛋白质-聚氨基酸偶联物的高效合成与应用

蛋白质-高分子偶联物是重要的临床药物,可用于多种疾病的治疗.寻找新的生物可降解高分子材料来替代传统的聚乙二醇和发展高效、位点特异性的偶联方法是该领域目前所面临的2个重要挑战.聚氨基酸是一类具有较好生物相容性、可生物降解、含有丰富侧链官能团的仿生功能高分子,在蛋白质修饰方面具有突出的优势,是有较大潜力的聚乙二醇替代物.本专论主要从新型α-氨基酸-N-羧基酸酐(Ncarboxyanhydrides,NCA)可控开环聚合方法、聚氨基酸原位官能化制备位点特异性蛋白质偶联物、扩展功能聚氨基酸分子库调控蛋白质功能等3个方面详细介绍蛋白质-聚氨基酸领域的研究进展,并对这类新型偶联物的发展进行了简单的评述和展望.     

非糖基化促红细胞生成素的聚乙二醇定点修饰及修饰产物性质

临床用重组人促红细胞生成素(rhEpo)是中国仓鼠卵巢细胞(Chinese hamster ovary cell, CHO)表达的糖蛋白, 糖基对稳定蛋白的结构和生物活性非常重要, 但CHO表达体系生产成本高、产量低. 以大肠杆菌表达的促红细胞生成素为非糖基化蛋白(rh-ngEpo), 对其进行聚乙二醇(PEG)修饰可以提高蛋白稳定性和体内循环半衰期. 本文采用分子量为20000的N-末端专一性的单甲氧基聚乙二醇-丙醛(mPEG-ALD)修饰rh-ngEpo, 对影响修饰反应的因素进行了考察. 结果表明, 在最佳反应条件下, 单修饰率可达55%. 修饰混合物经离子交换层析分离, 获得了纯度大于95%的单修饰产物, 其二、三级结构证明与原蛋白相似. 肽图分析结果表明, PEG绝大部分修饰在蛋白N-末端的氨基酸残基上. ELISA分析表明, 单修饰产物的体外活性虽然比修饰前减少30%, 但热稳定性得到显著增强, 在SD大鼠体内的药代动力学性质得到显著提高. 研究结果表明, PEG可以在一定程度上替代糖基的作用, PEG修饰的非糖基化Epo有望成为一种新型的促红细胞生成蛋白药物.     

功能化金属-有机框架材料在肿瘤治疗中的研究进展

恶性肿瘤由于其易转移、复发等特点，已经严重危害到人类的生命健康.近年来，研究人员设计了大量纳米药物载体，将抗肿瘤药物安全有效地运载到肿瘤，有效地提高了药效并降低了毒副作用.金属有机框架材料（metal-organic frameworks，MOFs）是一类有序、多孔的晶态材料，具有比表面积大、结构可设计性强、易生物降解等独特优势，已经被广泛应用于气体吸附与分离、催化、药物传递、生物大分子固载以及肿瘤治疗等方面.目前，基于MOFs的生物医用研究主要集中在MOF材料的可控合成，表面修饰，基于MOF独特理化性质发展的多模式成像技术以及肿瘤靶向的药物运载技术等几个方面.主要介绍了基于MOFs构建的生物功能化材料在肿瘤治疗中的应用，并对其在生物医学领域的应用进行了展望.     

聚乙二醇和叶酸对层状双金属氢氧化物颗粒的表面修饰

以具有伪装隐形作用的聚乙二醇(PEG)和具有靶向作用的叶酸(FA)为修饰剂, 以氨丙基三甲氧基硅烷(APTMS)为连接剂, 对Mg₃Al-NO₃层状双金属氢氧化物(LDH)进行了表面修饰, 制备了LDH-PEG-FA纳米颗粒, 并通过X射线衍射(XRD)、 透射电子显微镜(TEM)、 紫外-可见光谱(UV-Vis)、 傅里叶变换红外光谱(FTIR)、 粒度分布分析和元素分析等技术对其结构进行了表征. 结果表明, PEG和FA的修饰量可由其原料配比调控, 修饰后的产物具有良好的水再分散性, 这主要源于修饰层的空间位阻效应. 预期LDH-PEG-FA同时具有伪装隐形性和靶向性, 可用于药物载体等领域.     

两相体系检测聚乙二醇化粒细胞集落刺激因子的修饰度

研究了温度、平衡时间等对硫氰铁铵-氯仿两相体系检测不同分子量聚乙二醇(PEG)的影响因素,优化检测条件是温度30℃、平衡时间2h,检测限是6.65μg.利用PEG(30000)修饰粒细胞集落刺激因子(GCSF),分离纯化得到纯度大于97%的单修饰PEG30k-GCSF,建立了检测其修饰度的方法,得出PEG修饰度为19.4%,与理论值(20%)接近.此法能准确检测蛋白质或多肽的聚乙二醇修饰度.     

Advances and trends in the design, analysis, and characterization of polymer–protein conjugates for “PEGylaided” bioprocesses

In addition to their use as therapeutics and because of their enhanced properties, PEGylated proteins have potential application in fields such as bioprocessing. However, the use of PEGylated conjugates to improve the performance of bioprocess has not been widely explored. This limited additional industrial use of PEG-protein conjugates can be attributed to the fact that PEGylation reactions, separation of the products, and final characterization of the structure and activity of the resulting species are not trivial tasks. The development of bioprocessing operations based on PEGylated proteins relies heavily in the use of analytical tools that must sometimes be adapted from the strategies used in pharmaceutical conjugate development. For instance, to evaluate conjugate performance in bioprocessing operations, both chromatographic and non-chromatographic steps must be used to separate and quantify the resulting reaction species. Characterization of the conjugates by mass spectrometry, circular dichroism, and specific activity assays, among other adapted techniques, is then required to evaluate the feasibility of using the conjugates in any operation. Correct selection of the technical and analytical methods in each of the steps from design of the PEGylation reaction to its final engineering application will ensure success in implementing a "PEGylaided" process. In this context, the objective of this review is to describe technological and analytical trends in developing successful applications of PEGylated conjugates in bioprocesses and to describe potential fields in which these proteins can be exploited.     

Characterization of Electrospun Polysuccinimide-Dopamine Conjugates and Effect on Cell Viability and Uptake

Biocompatible nanofibrous systems made by electrospinning have been studied widely for pharmaceutical applications since they have a high specific surface and the capability to make the entrapped drug molecule amorphous, which increases bioavailability. By covalently conjugating drugs onto polymers, the degradation of the drug as well as the fast clearance from the circulation can be avoided. Although covalent polymer–drug conjugates have a lot of advantages, there is a lack of research focusing on their nano-formulation by electrospinning. In this study, polysuccinimide (PSI) based electrospun fibrous meshes conjugated with dopamine (DA) are prepared. Fiber diameter, mechanical properties, dissolution kinetics and membrane permeability are thoroughly investigated, as these are crucial for drug delivery and implantation. Dopamine release kinetics prove the prolonged release that influenced the viability and morphology of periodontal ligament stem cells (PDLSCs) and SH-SY5Y cells. The presence of dopamine receptors on both cell types is also demonstrated and the uptake of the conjugates is measured. According to flow cytometry analysis, the conjugates are internalized by both cell types, which is influenced by the chemical structure and physical properties. In conclusion, electrospinning of PSI-DA conjugates alters release kinetics, meanwhile, conjugated dopamine can play a key role in cellular uptake.     

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.     

Polymer therapeutics: concepts and applications

Polymer therapeutics encompass polymer-protein conjugates, drug-polymer conjugates, and supramolecular drug-delivery systems. Numerous polymer-protein conjugates with improved stability and pharmacokinetic properties have been developed, for example, by anchoring enzymes or biologically relevant proteins to polyethylene glycol components (PEGylation). Several polymer-protein conjugates have received market approval, for example the PEGylated form of adenosine deaminase. Coupling low-molecular-weight anticancer drugs to high-molecular-weight polymers through a cleavable linker is an effective method for improving the therapeutic index of clinically established agents, and the first candidates have been evaluated in clinical trials, including, N-(2-hydroxypropyl)methacrylamide conjugates of doxorubicin, camptothecin, paclitaxel, and platinum(II) complexes. Another class of polymer therapeutics are drug-delivery systems based on well-defined multivalent and dendritic polymers. These include polyanionic polymers for the inhibition of virus attachment, polycationic complexes with DNA or RNA (polyplexes), and dendritic core-shell architectures for the encapsulation of drugs. In this Review an overview of polymer therapeutics is presented with a focus on concepts and examples that characterize the salient features of the drug-delivery systems.     

Native PAGE eliminates the problem of PEG-SDS interaction in SDS-PAGE and provides an alternative to HPLC in characterization of protein PEGylation

PEGylation of proteins has become an increasingly important technology in recent years. However, determination and characterization of the PEGylation products are problematic especially for the reaction mixture containing various modified proteins, unreacted PEG, and unmodified protein. A comparative study was carried out with two HPLC methods and two electrophoresis methods for characterization of the reaction mixture in PEGylation of HSA with PEG 5000, 10000, and 20000. RP-HPLC fails to give the correct information about the reaction of PEG 20000. Size-exclusion HPLC (SE-HPLC) produced very poor resolution on the PEG 5000 reaction. SDS-PAGE can run multiple samples of all PEGylation but the bands were smeared or broadened probably due to the interaction between PEG and SDS. On the other hand, native PAGE eliminates the problem of PEG-SDS interaction and provides better resolutions for all samples. Various PEGylated products and unmodified protein migrate differentially in native PAGE under nondenatured conditions. The results demonstrated that native PAGE could be a good alternative to HPLC and SDS-PAGE for the analysis of PEG-protein conjugates especially for characterization of the PEGylation mixture.     

Covalently cross-linked proteins & polysaccharides: Formation,characterisation and potential applications

This review presents recent research conducted on the development of various protein-polysaccharide conjugates, their functional properties and industrial applications. These conjugates are formed by the glycosylation of food proteins with carbohydrates via the Maillard reaction and are capable of improving the functional properties of proteins. The Maillard reaction facilitates covalent bonding between a reducing group of a carbohydrate and an amino group of a protein under controlled conditions of temperature, time, pH, and relative humidity. There is a great deal of interest in modifying the functional properties of proteins and in the use of novel conjugates for various industrial applications. This review discusses various methods and their implications for preparing and characterising these conjugates. Furthermore, the physicochemical properties of conjugates such as solubility, thermal stability, emulsifying activity, emulsion stabilising properties, gelling and foaming properties are also analysed. A novel processing technology, a spinning disc reactor, could be an alternative process for the production of protein–polysaccharide conjugates, with desirable functionality in different food systems.     

基于金纳米粒子的光学生物传感方法研究进展

光学生物传感是以产生各种光学信号为检测基础的一种微量分析技术,具有操作简便、检测成本低、抗干扰能力强及可实现原位检测等优点,在临床诊断、药物分析、环境监测等领域显示出广阔的应用前景.作为纳米材料重要成员之一的金纳米粒子(AuNPs),因其独特的光学性质被广泛用于光学生物传感方法的构建.该文综述了近年来基于金纳米粒子的光...     

Adsorption of poly(ethylene glycol)-modified lysozyme to silica

Covalent grafting of poly(ethylene glycol) (PEG) to pharmaceutical proteins, "PEGylation", is becoming more commonplace due to improved therapeutic efficacy. As these conjugates encounter interfaces in manufacture, purification, and end use and adsorption to these interfaces may alter achievable production yields and in vivo efficacies, it is important to understand how PEGylation affects protein adsorption mechanisms. To this end, we have studied the adsorption of unmodified and PEGylated chicken egg lysozyme to silica, using optical reflectometry, total internal reflection fluorescence (TIRF) spectroscopy, and atomic force microscopy (AFM) under varying conditions of ionic strength and extent of PEG modification. PEGylation of lysozyme changes the shape of the adsorption isotherm and alters the preferred orientation of lysozyme on the surface. There is an abrupt transition in the isotherm from low to high surface excess concentrations that correlates with a change in orientation of mono-PEGylated conjugates lying with the long axis parallel to the silica surface to an orientation with the long axis oriented perpendicular to the surface. No sharp transition is observed in the adsorption isotherm for di-PEGylated lysozyme within the range of concentrations examined. The net effect of PEGylation is to decrease the number of protein molecules per unit area relative to the adsorption of unmodified lysozyme, even under conditions where the surface is densely packed with conjugates. This is due to the area sterically excluded by the PEG grafts. The other major effect of PEGylation is to make conjugate adsorption significantly less irreversible than unmodified lysozyme adsorption.     

Regulation of Catalytic Activity of Recombinant L-Asparaginase from <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis> by Conjugation with a PEG-Chitosan Copolymer

A new approach for the regulation of catalytic properties of the medically significant enzyme L-asparaginase is suggested based on the formation of conjugates with PEG-chitosan (chitoPEGylation). The efficiency of this approach is demonstrated using recombinant L-asparaginase from Rhodospirillum rubrum (RrA). This preparation is immunologically different from the one used in medical practice preparations of L-asparaginase from E. coli, which offers a promising alternative for applications in the case of hypersentsitivity development. The low level of activity of RrA towards L-glutamine, which decreases significantly the chance of side effects developing, is an advantage of RrA. The technique for the synthesis of the RrA conjugates with PEG-chitosan (chitoPEGylation) of a varying modification degree is developed. It is established that conjugation of RrA with PEG-chitosan increased the specific activity of the enzyme in comparison with the native one. The activity changes from 56 IU/mg (for the native enzyme) to 61–72 IU/mg (for the conjugates) depending on the degree of chitosan PEGylation. The secondary structure of the Rhodospirillum rubrum asparaginase conjugates with PEG-chitosan is examined using CD- and IR-spectroscopy. It is found that the enzyme structure changed only slightly as a result of conjugation with PEG-chitosan: the content of α-helices changed from 36% (for the native enzyme) to 30–33% (for the conjugates). The content of β-structures changed from 15% (for the native enzyme) to 18% (for the conjugate). The obtained data open new opportunities for the synthesis of L-asparaginase preparations with improved biocatalytic properties.     

Synthesis of degradable functional poly(ethylene glycol) analogs as versatile drug delivery carriers

Poly(ethylene glycol) (PEG) is widely used as a water soluble carrier for polymer-drug conjugates. Herein, we report degradable linear PEG analogs (DPEGs) carrying multifunctional groups. The DPEGs were synthesized by a Michael addition based condensation polymerization of dithiols and PEG diacrylates (PEGDA) or dimethacrylates (PEGDMA). They were stable at pH 7.4 but quickly degraded at pH 6.0 and 5.0. Thus, DPEGs could be used as drug carriers without concern for their retention in the body. DPEGs could be made to carry such functional groups as terminal thiol or (meth)acrylate and pendant hydroxyl groups. The functional groups were used for conjugation of drugs and targeting groups. This new type of PEG analog will be useful for drug delivery and the PEGylation of biomolecules and colloidal particles.