单修饰重组水蛭素的设计,制备及体外生物活性测定

Hirudin, the most potent inhibitor of thrombin found in nature, has a short half-life in serum, which sig-nificantly limits its clinical application as an anticoagulant. Recently, PEGylation has been commonly used as an effective method to prolong its half-life in serum. In contrast to the nonspecific pEGylation under basic conditions that targets lysine residues randomly, PEGylation sites under mildly acidic conditions preferably targets histidine residues, and there is only one histidine residue at 51 in r-hirudin; therefore, succinimidyl carbonyl methoxy poly-ethylene glycol （SC-mPEG, 20000） was attached to r-hirudin at mildly acidic pH to favor the formation of mono-PEGylated r-hirudin. The reaction mixture with high mono-PEGylated ratio was easily separated by a one-step ion-exchange chromatographic （IEC） procedure. Approximately 79.71% of the mono-PEGylated r-hirudin was PEGylated at His51, which showed that the acidic PEGylation operation prevented the PEGylation of active center （Lys47） of r-hirudin in pdnciple. Mono-PEGylated.product with purity higher than. 95% was obtained as the pre-dominant product, and 34% of the anticoagulant activity was retained in vitro. The staining method for sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） was improved to obtain perfect electrophoretic pattern in less than 5min. More accurate molecular weight was deduced due to the use of PEGs as molecular weight standards.     

甲基纤维素湿敏水凝胶中溶菌酶微球性质的体外评价

Long-terrn injectable microspheres have some inherent disadvantages such as migration of microspheres from the originalsite an.d the burst effect. In order to avoid these problems, microsphere-loaded thermosensitive, hydrogel system was designed and expected to achieve a zero-order release Of biomolecular drugs in relativehigh initial drug loadings. Lysozyme, an antibacterial protein usually used to reduce prosthetic valve endocarditis,was selected as the model drug. Poly （DL-lactide-co-glycolide） （PLGA） microspheres, prepared by solvent evaporation method, were employee to encapsulate lysozyme and dispersed into thermosensitive pre-gel solution containing methylcellulose （MC）, polyethylene glycol （PEG）, sodium citrate （SC）, and sodium alginate （SA）. The mixture could act asadrug reservoir by.performing sol-gel transition rapidly if the temperature was raised from roomtemperature to 37℃. The in vitro release results showed that the burst effect was avoided due to strengthening ofdiffusion resistance in the gel. The formulation was able.to deliver lysozy.me for over.30 daysin a nearly zero-order release profile with a rate of 32.8μg.d^-1 which exhibits its remarkable potential for effective aoolication in long-term drug delivery.     

固定化金属离子亲和分配用的亚胺二乙酸-聚乙二醇偶联物的制备

The synthesis route was investigated and optimized for the preparation of iminodiacetic acid polyethylene glycol (IDA-PEG) for immobilized metal ion affinity partitioning in aqueous two-phaze systems. IDA PEG was synthesized from PEG in two steps by the reaction of iminodiacetic acid with a monosubetituted derivative of epichlorohydrin-activated PEG. The Cu2+ content combined with IDA-PEG was determined by atomic absorp tion spectrometry as 0.5 mol.mol-1 (PEG). Furthermore, the affinity partitioning behavior of lactate dehydrogenase in polyethylene glycol/hydroxypropyl starch aqueous two-phaze systems was studied to clarify the affinity effect of the Cu(Ⅱ)-IDA-PEG.     

载三氧化二砷的PEG—PLGA隐性纳米粒的制备及体外研究

The aim of this study was to prepare arsenic trioxide （ATO）-loaded stealth PEGylated PLGA nanoparticles （PEG-PLGA-NPs） and to assess the merits of PEG-PLGA-NPs as drug carriers for ATO delivery. PEG-PLGA copolymer was synthesized with methoxypolyethyleneglycol （Mw=5000）, D, L-lactide, and glycolide by the ring-opening polymerization method. Amorphous ATO was transformed into cubic crystal form to increase its solu-bility in the organic solvent. ATO-loaded PEG-PLGA-NPs were prepared by the modified spontaneous emulsification solvent diffusion （SESD） method, and the main experimental factors influencing the characteristics of nanopar- ticles were investigated, to optimize the preparation. To confirm the escape of PEG-PLGA-NPs from phagocytosis by phagocytes, PEG-PLGA-NPs labeled rhodamine B uptake by murine peritoneal macrophages （MPM） were analyzed by flow cytometry. The results showed that the physicochemical characteristics of PEG-PLGA-NPs were affected by the type and concentration of the emulsifiers, polymer concentration, and drug concentration. ATO-loaded PEG-PLGA-NPs, with particle size of 120.8nm, zeta potential of-10.73mV, encapsulation efficiency of 73.6%, and drug loading of 1.36%, were prepared under optimal conditions. The images of transmission electron micros-copy （TEM） indicated that the optimized nanoparticles were near spherical and without aggregation or adhesion. The release experiments in vitro showed the ATO release from PEG-PLGA-NPs exhibited consequently sustained release for more than 26d, which was in accordance with Higuchi equation. The uptake of PEG-PLGA-NPs by MPM was found to decrease markedly compared to PLGA-NPs. The experimental results showed that PEG-PLGA-NPs were potential nano drug delivery carriers for ATO.     

Synthesis of texture-excellent mesoporous alumina using PEG1000 as structure-directing agent

By varying concentration of PEG1000 as a structure-directing agent,mesoporous alumina with excellent textural properties was synthesized.The prepared mesoporous alumina displays high thermal stability,as shown by its textural properties at different calcination temperatures of 600-850 °C.Characterization by SEM and TEM revealed that the added PEG surfactant induced the formation of petal-like alumina.XRD results clarified that all samples were amorphous and their peaks were around the peaks of γ-alumina.N_2 adsorption-desorption analysis showed that the prepared mesoporous alumina,if with PEG1000 in hydrolysis of aluminum isopropoxide,had excellent textural properties with large specific surface area,high pore volume and suitable pore size.The petal-like structure existing in the alumina samples improved their textural parameters,and the role and influential mechanism of PEG1000 were analyzed.     

Mesoporous tablet-shaped potato starch aerogels for loading and release of the poorly water-soluble drug celecoxib

In this study, after determination of the optimal values of the effective parameters in the synthesis using experimental design software, tablet-shaped potato starch aerogels were synthesized at the optimal condition in order to be used as a drug carrier. The celecoxib, as the model drug, was loaded into the aerogel matrix during the solvent exchange step. FTIR(Fourier Transform Infrared Spectroscopy), FESEM and HRTEM(Transmission Electron Microscopy) analyses showed that celecoxib has been successfully loaded into aerogel matrix. Also, XRD analysis showed that most of the celecoxib has been loaded in amorphous form. In vitro studies were performed in both simulated gastric and intestinal fluids. The release kinetics showed that the loaded celecoxib dissolved faster than crystalline celecoxib. At rotational speed of 100 r·min~(–1), about 26% and 50% and at rotational speed of 50 r·min~(–1),about 20% and 42% drug was released during the first 30 min of soaking in the simulated gastric fluid and simulated intestinal fluid, respectively. The release of the mentioned drug was increased up to 60% and 98% at a rotational speed of 100 r·min~(–1) and up to 46% and 93% at a rotational speed of 50 r·min~(–1) at the end of 5 h in the simulated gastric fluid and simulated intestinal fluid, respectively. It could be concluded that potato starch aerogels can be very useful in many drug delivery applications along with conventional micronization techniques. Modeling of release data showed that the release kinetics follows the Korsmeyer Peppas model, which considers phenomena of matrix erosion and drug diffusion.     

缩放管内带衰减性自旋流的局部性能

The distributions of thermal resistance in viscous sublayer,buffer region and turbulent core region,local flow resistance and local heat transfer characteristics at different locations downstream of the twisted-tape element were numerically studied in a converging-diverging tube inserted with spaced twisted-tape element by analyzing the attenuation of self-sustaining swirl flow.The results showed that the local performance was poor as thermal resistance was too concentrated in its distribution for a particular region.The more uniform the distribution of thermal resistance,the better the local performance.The local performance reached its best when the fluid just left the spaced twisted-tape,in which the flow resistance dropped substantially but the enhancement of heat transfer was still significant.The self-sustaining flow was maintained at a long distance.The best performance was at the length of 36.85 times the diameter,increasing by 6.8% compared with a converging-diverging tube.     

反应沉淀法制备超细硫酸沙丁胺醇颗粒及其粉雾剂的表征

The preparation of ultra-fine particles of salbutamol sulphate （SS） was accomplished with a reactive precipitation pathway, in which salbutamol and sulphuric acid were Used as reactants with the solvents of ethanol.The effects of sulphuric acid concentration, reaction temperature, stirring rate, and reaction time onthesize of the particle were investigated. A binary mixture composed of lactose and SS was prepared to evaluate SS. The results showed that ultra-fine SS particles with controlled diameters ranging between 3 μm and 0.8 μm and with a narrow distribution could be achieved. The morphology consisting of clubbed particles wassuccess.fully obtained. The purity of the particles reached above 98% with-UV detection. The dose- of dry powder inhalation was obtained by blending the particles with recrystallized lactose, which acted as a carrier. The deposition quantity of the drug in breathing tract was estimated using a twin imPinger apparatus. Compared with the Shapuer powder （purchased in the market）, the results showed that SS_particles had more quantifies.subsided in simulative lung.. _     

椰油丙基酰胺甜菜碱/聚乙二醇水溶液的分相研究

Phase separation behavior of cocamidopropyl betaine/water/polyethylene glycol (PEG) system was studied. The effects of concentration and molecular weight of PEG on the phase separation behavior were investigated. Clouding occurred when the con-centration of PEG was large enough in the betaine aqueous solution, and the concentration of PEG at cloud point decreased with the increase of PEG molecular weight for a constant betaine concentration. The bottom phase was the PEG-rich phase, and the upper phase was the betaine-rich phase. The volumetric ratio of PEG-rich phase to betaine-rich phase, at the same difference between the PEG concentration and the one at the cloud point, Ccp (0.1 g•ml－1), decreased as the PEG molecular weight increased and approached 1 for higher PEG molecular weight (about 20000), which was similar to the typical aqueous two-phase system. This volumetric ratio depended on the initial PEG concentration, but independent of PEG molecular weight. The concentration ratio of betaine to PEG in both phases depended on the Ccp, independent of PEG molecular weight.     

低含液率多相管路平均持液率

The experimental study on average holdup in multiphase pipelines with low loads of liquids was conducted on a large multiphase flow loop. The average holdup increased with increasing liquid load and decreased with increasing gas velocity, and also depended on the undulation of pipeline and flow patterns in the pipeline. The effects of eight dimensionless parameters on average holdup were seriated with grey relational degree analysis so as to find the most important factors affecting the average holdup in multiphase pipelines with low loads of liquids. An average holdup correlation based on low liquid loads was also developed and the correlation related the most important three factors including superficial gas velocity number (Ngw), superficial liquid velocity number (Nlw) and liquid Reynolds number (Rel) to the average holdup. Finally the correlation was tested with the field data and the result was satisfying.     

Current advances in the non‐chromatographic fractionation and characterization of PEGylated proteins

For more than 30 years, PEGylation has been used to improve the physicochemical properties of several proteins and therapeutic drugs having a major impact in the biopharmaceutical industry. The purification of PEGylated proteins usually involves two basic challenges: (1) the separation of PEG‐proteins from other reaction products; and (2) the sub‐fractionation of PEG‐proteins on the basis of their degree of PEGylation and positional isomerism. Currently, most PEGylated protein purification processes are based on chromatographic techniques, especially size exclusion chromatography (SEC) and ion exchange chromatography (IEX). Nonetheless, other less frequently used strategies based on non‐chromatographic techniques such as ultrafiltration, electrophoresis, capillary electrophoresis, and aqueous two‐phase systems have been developed in order to fractionate and analyze PEGylated derivates. This review presents current advances in some of the most widely used non‐chromatographic strategies for the fractionation and analysis of PEG‐protein conjugates. Copyright © 2010 Society of Chemical Industry     

PEG-proteins: Reaction engineering and separation issues

Poly(ethylene glycol)-conjugated (or PEGylated) proteins are an increasingly important class of therapeutic proteins that offer improved in vivo circulation half lives over their corresponding native forms. Their production involves covalent attachment of one or more poly(ethylene glycol) molecules to a native protein, followed by purification. Because of the extremely high costs involved in producing native therapeutic proteins it is important that subsequent PEGylation processes are as efficient as possible. In this paper, reaction engineering and purification issues for PEGylated proteins are reviewed. Paramount considerations for PEGylation reactions are specificity with respect to the conjugation site and overall yield. Batch PEGylation reaction methods are discussed, along with innovative methods using packed bed or “on-column” approaches to improve specificity and yield. Purification methods are currently dominated by ion exchange and size exclusion chromatography. Other methods in common use for protein separations, including hydrophobic interaction chromatography, affinity chromatography and membrane separations, are rarely used in PEGylated protein purification schemes. A better understanding of the effects of PEGylation on the physicochemical properties of proteins (isoelectric point, surface charge density and distribution, molecular size and relative hydrophobicity) and interactions between PEGylated proteins and surfaces is needed for the future development of optimal purification processes and media.     

Impacts of PEGylation on the gene and oligonucleotide delivery system

Poly(ethylene glycol) (PEG) is the most widely used polymer and also the gold standard in the field of drug delivery. Therapeutic oligonucleotides, for example, are modified with PEG at the terminus to increases nuclease resistance and the circulating half‐lives. The surface of nanoparticle such as micelle and liposome has been also modified with PEG. At present, one PEGylated therapeutic oligonucleotide has been approved for the market and several more PEGylated products including oligonucleotide and liposome are being tested in clinical settings. This review summarizes the methods and effects of PEGylation on gene delivery. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40293.     

Synthesis and Chromatographic Separation of Monomethoxypolyethylene Glycol Modified Insulin

Abstract

Insulin was modified with monomethoxypolyethylene glycol (MPEG)‐succinimidyl succinate and succinimidyl ester of carboxymethyl MPEG. Effects of reaction solvents, initial molar ratio of MPEG derivative to insulin and reaction time on PEGylation of insulin were investigated by 2,4,6‐trinitrobenzenesulfonic acid spectrophotometric assay and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Sephadex G75 size exclusion chromatography (SEC), ion exchange chromatography (IEC) and reversed phase‐high performance liquid chromatography (RP‐HPLC) were applied to separate PEGylated insulin. IEC and RP‐HPLC were proved to be efficient tools on separation of different PEGylated insulin species.     

Synthesis and selected properties of polyurethanes with monodisperse hard segments based on hexane diisocyanate and three types of chain extenders

Poly(ethylene glycol) grafted octadecyl quaternized carboxymethyl chitosan (PEG-g-OQC) copolymers were synthesized to both improve the biocompatibility of OQC and form PEGylated cationic polymeric liposomes (CPLs), which composed of the mixture of OQC, cholesterol, and PEG-g-OQC. Structure of the copolymers was characterized by using Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectroscopy (¹H-NMR), and X-ray diffraction (XRD). The methyl tetrazolium (MTT) assay with L929 cell lines confirmed that PEGylation can decrease the cytotoxicity of OQC. PEGylated CPL nanoparticles (NPs) can be prepared by adding different weight ratio of PEG-g-OQC in the mixture. Paclitaxel was successfully incorporated into PEGylated CPLs with high drug encapsulating efficiency (>90%) and drug loading capacity (>15%). Physical stability experiment showed that paclitaxel-loaded PEGylated CPLs was stable with little change of particle size and size distribution in the condition of freeze-dried by adding mannitol or in high temperature and high pressure. Power or reagent of drug-loaded PEGylated CPLs showed a slow steady release profile for paclitaxel. These results show that PEG-g-OQC and CPLs have potential application as a drug delivery vehicle. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

PEGylation of deuterohaemin–alanine–histidine–threonine–valine–glutamic acid–lysine and its influence on activity,stability, and aggregation

Deuterohaemin–alanine–histidine–threonine–valine–glutamic acid–lysine (DhHP‐6) is a synthetic heme‐containing peroxidase mimic that exhibits a high peroxidase enzyme activity. Compared to other microperoxidases, DhHP‐6 has a poor stability and tends to aggregate in aqueous solutions. In this study, poly(ethylene glycol) (PEG) was used to improve the properties of DhHP‐6. Factors that affected the PEGylation product yield were investigated. PEGylated DhHP‐6 (mPEG–DhHP‐6) was characterized by reversed‐phase high‐pressure liquid chromatography (RP‐HPLC), matrix‐assisted laser desorption/ionization time of flight mass spectra (MALDI‐TOF‐MS), and ultraviolet–visible (UV–vis) spectroscopy. The results show that the optimal PEGylation reaction conditions were achieved when the PEGylation was conducted in a borate buffer solution at pH 8.0 and 25°C for 4 h with a feeding ratio of 2 equiv of active PEG. After PEGylation, mPEG–DhHP‐6 showed a great improvement in its stability with little activity loss. The UV–vis spectra of DhHP‐6 and mPEG–DhHP‐6 in different pH solutions showed that the aggregation of DhHP‐6 was partly suppressed after PEGylation. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

PEGylated chitosan derivatives: Synthesis, characterizations and pharmaceutical applications

This review sets out to describe and discuss the synthetic approaches and the fields of application of PEGylated chitosan copolymers especially for medical use. The PEGylation of chitosan and chitosan derivatives is able to add new physicochemical properties to the cationic polysaccharide polymers, thereby overcoming some limitations, especially regarding their solubility and their use in drug and gene delivery (DNA and siRNA). All methods of derivatization have been considered and described together with the different methods of characterization of the copolymers. The capacities of PEGylated chitosan to reduce chitosan toxicity, to enhance membrane permeation and to form thermosensitive hydrogels have also been discussed.     

水蛭素活性分析方法的研究

水蛭素是凝血酶的天然抑制剂,是一种很有前途的抗凝药物。水蛭素活性的测定对于水蛭素的得率和比活力非常重要,讨论了水蛭素活性测定的几种方法：凝血酶直接滴定法、Chromonym TH为生色底物的比色法和光散射法,以及凝血酶滴定法的改进。     

mPEG–NHS carbonates: Effect of alkyl spacers on the reactivity: Kinetic and mechanistic insights

Nowadays, the chemical conjugation to mPEG, also known as PEGylation, is a well-recognized technology used to improve the pharmaceutical properties of the therapeutic proteins. Over the last 20 years, more than 10 PEGylated macromolecules reached the market with tremendous success, whereas various other bioconjugates are under advanced clinical trials. mPEG–N-hydroxysuccinimidyl carbonate is an important reagent of widespread application for the PEGylation of biomacromolecules. One of the most important challenges in this technology is the development of more selective PEGylation reagents aimed to provide more consistent polymer–protein conjugates. One approach followed to improve the selectivity of PEGylation reagents is the design of less reactive derivatives, for example, by incorporation of alkyl spacers between the polymer chain and the terminal reactive group. In this work, we prepared a family of mPEG–N-hydroxysuccinimidyl carbonates bearing spacers of up to 6 carbon atoms. The kinetics of hydrolysis of the carbonates was studied under different experimental conditions, as a straight measure of the influence of the length of the spacer on the reactivity. By DFT calculations, we propose a detailed mechanism for the hydrolysis reaction. The influence of the length of alkyl spacer on the reactivity of the carbonates and related esters is studied and discussed in detail. Finally, to further evaluate the reactivity, selected N-hydroxysuccinimidyl carbonates were studied in the conjugation reaction of bovine lactoferrin. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47028.