Cellulose-based Polymeric Liquid Crystals as a Biomimetic Modifier for Suppressing Protein Adsorption

A novel biomimetic protein-resistant modifier based on cellulose-based polymeric liquid crystals was described（PLCs）. Two types of PLCs of propyl hydroxypropyl cellulose ester（PPC） and octyl hydroxypropyl cellulose ester（OPC） were prepared by esterification from hydroxypropyl cellulose, and then were mixed with polyvinyl chloride and polyurethane to obtain composite films by solution casting, respectively. The surface morphology of PLCs and their composite films were characterized by polarized optical microscopy（POM） and scanning electron microscopy（SEM）, suggesting the existence of microdomain separation with fingerprint texture in PLC composite films. Water contact angle measurement results indicated that hydrophilicity of PLC/polymer composite films was dependent on the type and content of PLC as well as the type of matrix due to their interaction. Using bovine serum albumin（BSA） as a model protein, protein adsorption results revealed that PLCs with protein-resistant property can obviously suppress protein adsorption on their composite films, probably due to their flexible LC state. Moreover, all PLCs and their composites exhibited non-toxicity by MTT assay, suggesting their safety for biomedical applications.

Protein Drug-Loaded Polymeric Nanoparticles

Considerable interest and research have focused on the administration of therapeutic proteins. For delivery of therapeutic proteins, bioavailability and stabilization of protein drugs to maintain therapeutically acceptable levels is an important challenge in clinical trials. To overcome these challenges, polymeric nanoparticles have become one of the best methods for protein delivery. In this review, we summarize the current available polymeric nanoparticles designed for protein delivery, current status, and advantages of protein delivery systems.

Transfer and Reactivity of Hydrogen Sulfide with Immobilized Hemeproteins in Polymeric Matrix

Hydrogen sulfide (H2S) has been related to be toxic and to have a role in human physiological functions. Therefore, there is a necessity to comprehend ways to scavenger hydrogen sulfide from different media. Here, we used recombinant metaquo-Hemoglobin I (metHbI) from Lucina pectinata and metaquo-myoglobin (metMb) encapsulated in the tetramethyl orthosilicate gel (TMOS), to facilitate the understanding of H2S transfer toward these metaquo-hemeproteins. In this sol-gel environment, metHbI binds and releases H2S with rate constants of 0.0597 M-1·s-1 and 6.67 × 10-5 s-1, respectively. The process generates an H2S affinity constant (kon/koff) of 8.9 × 102 M-1, which is 107 lowers than the analogous constant in solution (6.3 × 109 M-1). Although the H2S koff for the rHbI-H2S complex is almost similar with both sol-gel and solution. To further understand how the H2S koff from rHbI-H2S in solution (5 μM) is influenced by the protein concentration gradient, metHbI and metMb (25 μM) encapsulated in TMOS sol-gel. Under these circumstances, the H2S transfer from a solution of the rHbI-H2S complex to encapsulated hemeprotein resulted in koff values of 1.90 × 10-4 s-1, and 2.09 × 10-4 s-1 leading to the formation of rHbI-H2S and Mb-H2S species, respectively. The results suggest that the: 1) extreme ionic TMOS construct limits the H2S pathways to reach the hemeprotein active center, 2) possible interaction with metHbI hydrophilic forces increases the hydrogen bonding networking and decreases the H2S association constant, 3) hemeproteins concentration gradients between solution and sol-gels also influence its hydrogen sulfide transfer. In the presence of oxygen or hydrogen peroxide metMb generated a mixture of Mb-H2S and sulfmyoglobin derivative, while encapsulated metHbI reaction did not produce the sulfheme species. Consequently, the results show that metHbI encapsulated in TMOS is an excellent trap for H2S from solution or gas media.

Molecularly Imprinted Polymer Based on GO-QDs as Enhanced Fluorescent Nanoscale Device for Specific Recognition of Target Protein

In this work the enhanced molecularly imprinted optosensing material based on graphene oxide-quantum dots （ GO- QDs） was synthesized for highly selective and sensitive specific recognition of the target protein, bovine serum albumin （BSA）. Here, GO was introduced to enhance the efficiency of mass-transfer in recognition of target protein. Molecularly imprinted polymer coated GO-QDs using BSA as template （BMIP-coated GO-QDs ） exhibited a fast mass-transfer speed, which could be ascribed to the high volume of efficient surface area and high target recognition efficiency of the synthesized nanoscale device. Under optimal conditions, it was found that the BSA as target protein could remarkably quench the relative fluorescence intensity of BMIP- coated GO-QDs linearly in a concentration-dependent manner that was best described by a Stern-Volmer equation. The Ksv （Stern- Volmer constant） for template BSA was much higher than bovine hemoglobin （BHb） and lysozyme （Lyz）, implying a highly selective recognition ability of the BMIP-coated GO-QDs to BSA. This enhanced fluorescent nanoscale device may provide opportunities to develop a system that is efficient and effective and has potential in the design of highly effective fluorescent receptor for recognition of target protein in Droteomics studies.

A strong adhesive block polymer coating for antifouling of large molecular weight protein

Some proteins secreted by microorganisms have large molecular weights. We report here an approach to prepare coating by multilayer polymers for antifouling of proteins, especially the proteins with a large molecular weight.Stainless steel was used as the model substrate. The substrate was first coated with a hybrid polymer film, which was formed by simultaneous hydrolytic polycondensation of 3-aminopropyltriethoxysilane and polymerization of dopamine(HPAPD). After grafting the macroinitiator 2-bromoisobutyryl bromide, the block polymer brushes PMMA-b-PHEMA were grafted. Three proteins were used to test protein adsorption and antifouling behavior of the coating, including recombinant green fluorescent(54 k Da), recombinant R-transaminase(2 × 90 k Da), and recombinant catalase(4 × 98 k Da). It is demonstrated that the block polymer brushes not only can prevent the adsorption of small molecular weight proteins, but also can significantly reduce the adsorption of the large molecular weight proteins.

Electrochemical Impedance Analysis of Biofunctionalized Conducting Polymer-Modified Graphene-CNTs Nanocomposite for Protein Detection

We report an electrodeposited poly(pyrrole-co-pyrrolepropylic acid) copolymer modified electroactive graphene-carbon nanotubes composite deposited on a glassy carbon electrode to detect the protein antigen(cTnI). The copolymer provides pendant carboxyl groups for the site-specific covalent immobilization of protein antibody, antitroponin I. The hybrid nanocomposite was used as a transducer for biointerfacial impedance sensing for cTnI detection.The results show that the hybrid exhibits a pseudo capacitive behaviour with a maximum phase angle of 49° near 1 Hz,which is due to the inhomogeneous and porous structure of the hybrid composition. The constant phase element of copolymer is 0.61(n = 0.61), whereas, it is 0.88(n = 0.88) for the hybrid composites, indicating a comparatively homogeneous microstructure after biomolecular functionalization. The transducer shows a linear change in charge transfer characteristic(R_(et)) on cTnI immunoreaction for spiked human serum in the concentration range of 1.0 pg mL^(-1)–10.0 ng mL^(-1). The sensitivity of the transducer is 167.8 ± 14.2 Ω cm^2 per decade, and it also exhibits high specificity and good reproducibility.

牡蛎疱疹病毒结构蛋白真核表达系统构建及多聚化特性

牡蛎疱疹病毒(OsHV-1)在全球范围内导致牡蛎、扇贝与蚶类的大规模死亡,成为双壳贝类养殖产业的重要威胁。为了解OsHV-1的结构与致病机制。本研究利用人胚胎肾细胞(HEK293t),构建OsHV-1主要核衣壳蛋白(ORF104和ORF33)的真核表达系统,并对ORF104和ORF33潜在相互作用进行分析。实验首先通过特异性PCR扩增技术得到orf 104和orf 33的基因序列,根据其编码蛋白的理化性质、跨膜区与三维结构等生物信息学分析结果,选择pCDNA3.1(+)构建两种基因的重组表达质粒。重组质粒经大肠杆菌扩增、提取后,利用转染试剂Lipo8000™将pCDNA3.1(+)-orf 104与pCDNA3.1(+)-orf 33分别单独或共转染至HEK293t。然后,将转染后的细胞培养18 h后裂解收集蛋白。最后利用蛋白免疫印迹(Western blot,WB)与负染电镜检测两种目的蛋白的表达情况。结果显示,实验成功构建了OsHV-1衣壳蛋白ORF104和ORF33的重组表达质粒载体,通过真核细胞表达得到大小约为135与35 ku的目的蛋白。研究表明,表达质粒可在真核表达系统中实现蛋白单独转染与共转染,共转染蛋白间可能存在相互作用的趋势并形成多聚体,其中,ORF33自身即可形成分子质量不同的多聚体。本研究首次利用真核表达系统开展OsHV-1关键结构蛋白的表达,为进一步开展该病毒结构蛋白功能与互作,以及病毒入侵机制研究奠定基础。

不同外源Cd(Ⅱ)对Pseudomonas aeruginosa EPS的胁迫效应--产量、组分、吸附特性变化及其机制

以CdCl_(2)、CdSO_(4)和Cd(NO_(3))_(2)为胁迫因子,比较了不同阴离子在胁迫/诱导过程中对Pseudomonas aeruginosa(P.aeruginosa)胞外聚合物(EPS)的产量和特性的影响以及对Cd(II)吸附性能的变化.研究发现,当Cd(NO_(3))_(2)胁迫/诱导浓度为50mg/L时,EPS产量达到214.91mg/g VSS,其中蛋白质含量达到136.75mg/g VSS,较胁迫/诱导前增加了409.31%.此时EPS的吸附性能最好,对Cd(II)的平衡吸附量最大,较胁迫/诱导前增加了近一半.HPLC分析表明,EPS蛋白质中氨基酸的变化与EPS的吸附特性密切相关.3D-EEM、XPS和FTIR结果表明,EPS中C=O、C-N和N-H含量增加,有利于重金属的结合.竞争吸附实验发现,EPS对Cd(II)的亲和力高于Zn(II).

CMC-MMA-AA纳米乳液的制备及其抗蛋白质吸附性能的研究

本文以羧甲基纤维素(CMC)为乳化剂,甲基丙烯酸甲酯(MMA)和丙烯酸(AA)为单体,H_(2)O_(2)+Fe^(2+)为引发剂,采用乳液聚合技术制备了CMC-MMA-AA纳米乳液。通过SEM、DLS、TG、IR对乳胶粒的理化性质进行表征,并采用BLI、SDS-PAGE等方法验证纳米粒子的抗蛋白质吸附性能。结果表明:使用羧甲基纤维素包被后的聚甲基丙烯酸甲酯-丙烯酸纳米粒子粒径大小均一,粒径分布较窄,且具备良好的抗蛋白质吸附性能。

Effect of proteins,polysaccharides,and particle sizes on sludge dewaterability

Four batch experiments of hydrolysis and acidification were carried out to investigate the distributions of proteins （PN） and polysaccharides （PS） in the sludge, the PN/PS ratio, the particle sizes, and their relationship with sludge dewaterability （as determined by capillary suction time, CST）. The sludge flocs were stratified through centrifugation- and ultrasound-based method into four fractions： （1） slime, （2） loosely bound extracellular polymeric substances （LB-EPS）, （3） tightly bound EPS （TB-EPS）, and （4） pellet. The results showed that PN was mainly partitioned in the pellet （80.7%） and TB-EPS （9.6%） fractions, while PS distributed evenly in the four fractions. During hydrolysis and acidification, PN was transferred from the pellet and TB-EPS fractions to the slime fraction, but PS had no significant transfer trends. The mean particle sizes of the sludge flocs decreased with hydrolysis and acidification. The pH had a more significant influence on the dewaterability of sludge flocs than temperature. Sludge dewaterability during hydrolysis and acidification processes greatly deteriorated from 9.7 s at raw sludge to 340-450 s under alkaline conditions. However, it was just slightly increased under acidic conditions. Further investigation suggested that CST was affected by soluble PN, soluble PN/PS, and particle sizes of sludge flocs, but was affected slightly by total PN, PS, or PN/PS in the whole sludge flocs and other fractions （except slime）.

Design and Fabrication of Superparamaganitic Hybrid Microspheres for Protein Immobilization

Superparamagnetic poly（styrene）-co-poly（2-acrylanmido-2-methyl propanesulfonic acid） （PSt-co-PAMPS） and poly（methylmethacrylate）-co-poly（glycidyl methacrylate） （PMMA-co-PGMA） microspheres with mean size of 170 nm were prepared by emulsion polymerization in the presence of oleic acid-coated Fe3O4 nanoparticles. The structures, morphologies, diameter and diameter distribution of the as-prepared microspheres were identified by Fourier transform infrared spectroscopy （FT-IR） and transmission electron microscopy （TEM）. The saturation magnetizations of PSt-co-PAMPS and PMMA-co-PGMA microspheres are 21.94 and 25.07 emu/g, respectively. The as-synthesized magnetic microspheres were used for immobilization of Bovine serum albumin （BSA） by physical interaction and covalent interaction respectively. The equilibrium amount of BSA immobilized onto PMMA-co-PGMA microspheres was 86.48 mg/g microspheres in 90 min, while on PSt-co-PAMPS microspheres was 59.62 mg/g microspheres in 120 min.

Plasma graft of poly(ethylene glycol) methyl ether methacrylate (PEGMA) on RGP lens surface for reducing protein adsorption

Poly（ethylene glycol） methyl ether methacrylate（PEGMA） was grafted on fluorosilicone acrylate rigid gas permissible contact lens surface by means of argon plasma induced polymerization to improve surface hydrophilicity and reduce protein adsorption.The surface properties were characterized by contact angle measurement,x-ray photoelectron spectroscopy（XPS） and atomic force microscopy respectively.The surface protein adsorption was evaluated by lysozyme solution immersion and XPS analysis.The results indicated that a thin layer of PEGMA was successfully grafted.The surface hydrophilicity was bettered and surface free energy increased.The lysozyme adsorption on the lens surface was reduced greatly.

Interpenetrated Chitosan-Poly(Acrylic Acid-Co-Acrylamide) Hydrogels. Synthesis, Characterization and Sustained Protein Release Studies

Interpenetrated polymer networks of chitosan (CHI), polyacrylic acid (PAA) and polyacrylamide (PAM) were prepared by free radical polymerization. These hydrogels were either washed with double distilled water (CHI/PAA/PAM) A or hydrolyzed with 1M sodium hydroxide (NaOH), (CHI/PAA/PAM) S. Both types of hydrogels were characterized by infrared spectroscopy, microstructural techniques and compressive mechanical testing. Finally, hydrogels were loaded with bovine serum albumin (BSA) and release followed at different pHs. Infrared spectra analysis showed correspondence between hydrogels and monomer feed compositions. Hydrolyzed hydrogels, had increased water content and pH swelling dependence. Compression modulus of swelled hydrolyzed hydrogels decreased with increasing equilibrium water content. Higher BSA loadings were achieved on hydrolyzed hydrogels due to their high water content and porosity. Protein release from hydrogels was low (≤ 20% after 10 hours) at pH 1.2, but sustained release was observed at pH 6.8 and 7.4. The integrity of the protein released at 6.8 and 7.4 by hydrolyzed hydrogels was unaffected. The hydrogles showed no cytotoxic effects on human skin dermal fibroblasts as determined by MTT assay except for two compositions of (CHI/PAA/PAM) A samples, which after seven days presented a viability lower than 80% respect to the control.

Effects of Propofol combined with remifentanil on the levels of MBP,NSE and S100B protein,D-D and inflammatory factors in patients with acute craniocerebral trauma

Objective: To investigate the effects of Propofol combined with remifentanil on serum levels of MBP, NSE and S100B protein, D-D and inflammatory factors in patients with acute craniocerebral trauma. Methods: A total of 100 patients were selected with traumatic brain injury who underwent emergency surgery from August 2014 to May 2017 in our hospital, then randomly divided them into the control group and the experimental group, 50 cases each. The control group received isoflurane combined with remifentanil to maintain anesthesia, and the experimental group received propofol and remifentanil to maintain anesthesia. The inflammatory factors and the levels of MBP, NSE, S100B and D-D in the two groups before and after anesthesia (T0), 1H (T1) and postoperative 1H (T2) were detected and compared. Results: There was no significant difference between the two groups in the levels of TNF-α. The serum level of hs-CRP in two groups of T1, T2 increased significantly, the difference was statistically significant compared with T0, in the experimental group, serum level of hs-CRP at T1 and T2 was significantly higher than the control group, the difference was statistically significant. Conclusion: Propofol combined with remifentanil anesthesia for acute craniocerebral trauma can maintain the balance of inflammatory cytokine levels during the perioperative period, inhibit the elevation of serum MBP, NSE, S100B protein and D-D levels, reduce brain cell damage. It has a good protective effect on brain cells and is worthy of clinical application.

Polymer Nanoparticles Prepared by Supercritical Carbon Dioxide for in Vivo Anti-cancer Drug Delivery

A new approach for producing polymer nanoparticles made of bovine serum albumin-poly(methy methacrylate) conjugate by precipitating in supercritical CO2 is reported. The nanoparticles were loaded with the anti-tumor drug camptothecin. With albumin serving as a nutrient to cells, the drug-encapsulated nanoparticle shows an enhanced ability to kill cancer cells compared to that of the free drug in solution both in vitro and in vivo.

Engineered polymer nanoparticles incorporating L-amino acid groups as affinity reagents for fibrinogen

Synthetic polymer hydrogel nanoparticles(NPs)were developed to function as abiotic affinity reagents for fibrinogen.These NPs were made using both temperature-sensitive N-isopropyl acrylamide(NIPAm)and L-amino acid monomers.Five kinds of L-amino acids were acryloylated to obtain functional monomers:L-phenylalanine(Phe)and L-leucine(Leu)with hydrophobic side chains,L-glutamic acid(Glu)with negative charges,and L-lysine(Lys)and L-arginine(Arg)with positive charges.After incubating the NPs with fibrinogen,g-globulin,and human serum albumin(HSA)respectively,the NPs that incorporated Nacryloyl-Arg monomers(AArg@NPs)showed the strongest and most specific binding affinity to fibrinogen,when compared with g-globulin and HSA.Additionally,the fibrinogen-AArg binding model had the best docking scores,and this may be due to the interaction of positively charged AArg@NPs and the negatively charged fibrinogen D domain and the hydrophobic interaction between them.The specific adsorption of AArg@NPs to fibrinogen was also confirmed by the immunoprecipitation assay,as the AArg@NPs selectively trapped the fibrinogen from a human plasma protein mixture.AArg@NPs had a strong selectivity for,and specificity to,fibrinogen and may be developed as a potential human fibrinogen-specific affinity reagent.

Effect of Sludge Retention Time on the Fate of Proteins and Polysaccharides in AAO Process

Effect of sludge retention time( SRT) on the removal of potential and polysaccharides in an anaerobic / anoxic / aerobic( AAO) process was investigated. The Lowry method and anthrone method were used to detect proteins and polysaccharides. Total removal efficiency of proteins at SRT of 10 to 25 d in the AAO system was higher than 90%. Polysaccharides removal efficiencies were above 80% when SRT was increased from 15 to 25 d,whereas only 81% of polysaccharides was removed at SRT of 10 d. The biodegradation part of proteins and polysaccharides increased from87. 40% to 93% and from 74. 22% to 86. 94% with increasing SRTs.The ratios of polysaccharides and proteins in extracellular polymer substances( EPSs) were around 1. 5-3 in different SRTs. As SRT increasing,polysaccharides and proteins discharged with residual sludge decreased gradually. The amount of EPSs decreased with increasing SRTs.

Catechol-Based Polymers with High Efficacy in Cytosolic Protein Delivery

Polymers have been widely proposed as carriers for cytosolic protein delivery despite multiple barriers such as protein binding,cell internalization,and endosome escape during cytosolic delivery.Inspired by the strong binding affinity of natural polyphenols with proteins and cell membranes,herein we propose polyphenol modification to improve the efficacy of the protein delivery of cationic polymers.Catecholmodified dendrimers with balanced hydrophobic and hydrogen-bonding interactions show the highest efficacy for various cargo proteins and peptides while the pyrogallol-grafted ones exhibit the lowest efficacy due to increased ligand hydrophilicity.The catechol-based polymers efficiently deliver various bioactive proteins into the cytosol of live cells,exerting biofunctions after intracellular release,and successfully transmittingα-chymotrypsin into tumor cells in vivo to inhibit tumor growth.This study proves that polycatechols can serve as a family of highly efficient carriers for delivery of macromolecular biopharmaceuticals.

抗坏血酸/过氧化氢介导乳清分离蛋白与EGCG接枝聚合动力学

以抗坏血酸(Ascorbic Acid,Asc)和过氧化氢(Hydrogen Peroxide,H_(2)O_(2))为引发剂,研究乳清分离蛋白(Whey Isolate Protein,WPI)与表没食子儿茶素没食子酸酯((-)-Epigallocatechin Gallate,EGCG)接枝聚合动力学。系统研究活化时间、引发剂浓度、单体浓度和温度对接枝量的影响,通过测定反应级数和表观活化能,建立接枝反应动力学方程。结果表明,在Asc/H_(2)O_(2)体系中,由抗坏血酸自由基(Asc^(-)·)介导WPI与EGCG接枝反应。当反应条件为Asc浓度0.014mol/L,H_(2)O_(2)浓度0.10mol/L,溶液pH值6,温度308.15K以及活化时间120min时,接枝量为42.08mg/g。基于上述结果,我们可以得到在Asc浓度0.01~0.022mol/L,H_(2)O_(2)浓度0.09~0.12mol/L,EGCG浓度0.0012~0.0042mol/L,温度298.15~308.15K变化范围内,建立了WPI接枝EGCG的聚合速率方程,聚合反应的表观活化能E=12.96kJ/mol。研究结果为蛋白质的结构设计和可控制备提供理论指导,拓宽蛋白质在食品工业中的应用。

Evaluation of the secondary structures of protein in the extracellular polymeric substances extracted from activated sludge by different methods

The changes of protein secondary structures in the extracellular polymeric substances(EPS) extracted from activated sludge by four different methods were studied by analyzing the amide I region(1700–1600 cm-1) of the Fourier transform infrared spectra and model protein test. The results showed the molecular weight distribution of organic matter extracted by centrifugation, heating and cation exchange resin(CER) was similar, while the EPS extracted by centrifugation(Control) and CER had similar fluorescent organic matter. The protein secondary structures of extracted EPS by the four methods were different. The similarities of protein secondary structures between the EPS extracted by CER with the Control were the highest among the four extracted EPS. Although the EPS yield extracted by formaldehyde + NaOH method were the highest, its protein secondary structures had the lowest similarity with those extracted by the Control. Additionally, the effects of centrifugation and CER extraction on the secondary structures of bovine serum albumin were also lower than that of other extraction processes. CER enables the second maximum extraction of EPS and maximum retention of the original secondary structure of proteins.