Pentafluorophenyl ester-functionalized phosphorylcholine polymers: preparation of linear, two-arm, and grafted polymer-protein conjugates

Novel pentafluorophenyl (PFP)-ester-functionalized phosphorylcholine (PC) polymers of different architectures were prepared and conjugated to lysozyme as a model protein. Linear and two-arm poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC) structures containing PFP functionality at the chain-end were prepared by atom transfer radical polymerization (ATRP) from novel initiators. Additional conjugates were prepared from phosphorylcholine-substituted cyclooctene (PC-COE) polymers containing PFP-ester bearing comonomers. The polymer-protein conjugates were characterized by HPLC, FPLC, and DLS and were seen to retain most (~80% or greater) of their native enzymatic activity. Pharmacokinetic profiles of the polymer-protein conjugates were studied in mice and found to increase the circulation half-life compared with lysozyme alone.     

Starburst Conjugates of Proteins with Carbon-Chain Polymers Containing Low Molecular Biologically Active Compounds: Synthesis and Immunogenicity

The synthesis of starburst polymer–protein conjugates on the basis of bovine serum albumin and horseradish peroxidase was performed with the aim to study the possibilities of regulation of the immune response against the components of the conjugates. These polymers had one-point binding between the protein and the modifying carbon-chain polymer that contained 1-vinyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (a salsolinol analogue) or bradykinin (peptide hormone) residues in its carbon chain. Changes in the chemical nature of the carbon-chain part of the polymer–protein conjugate were shown to increase or decrease the level of antibody production both against the low-molecular compounds attached to the polymeric fragments and against the protein carrier.     

End-functionalized phosphorylcholine methacrylates and their use in protein conjugation

Polymer-protein conjugation was performed using N-hydroxysuccinimide and aldehyde-terminated zwitterionic polymers, and the resulting polymer-protein conjugates were characterized by gel electrophoresis and fast protein liquid chromatography. Methacryloyloxyethyl phosphorylcholine (MPC) polymers were prepared by atom transfer radical polymerization in which the requisite functional end-groups for protein conjugation were embedded within the polymerization initiators. These phosphorylcholine polymers were conjugated to lysozyme as a model protein, as well as two therapeutic proteins, granulocyte colony stimulating factor (G-CSF) and erythropoietin (EPO). These MPC polymer-protein conjugates represent alternatives to PEGylated proteins, with the potential to provide improved efficacy in a therapeutic treatment relative to the protein itself.     

Polymer conjugates: nanosized medicines for treating cancer

Interdisciplinary research at the interface of polymer chemistry and the biomedical sciences has produced the first polymer-based nanomedicines for the diagnosis and treatment of cancer. These water-soluble hybrid constructs, designed for intravenous administration, fall into two main categories: polymer-protein conjugates or polymer-drug conjugates. Polymer conjugation to proteins reduces immunogenicity, prolongs plasma half-life and enhances protein stability. Polymer-drug conjugation promotes tumor targeting through the enhanced permeability and retention (EPR) effect and, at the cellular level following endocytic capture, allows lysosomotropic drug delivery. The successful clinical application of polymer-protein conjugates (PEGylated enzymes and cytokines) and promising results arising from clinical trials with polymer-bound chemotherapy (e.g. doxorubicin, paclitaxel, camptothecins) has provided a firm foundation for more sophisticated second-generation constructs that deliver the newly emerging target-directed anticancer agents (e.g. modulators of the cell cycle, signal transduction inhibitors and antiangiogenic drugs) in addition to polymer-drug combinations (e.g. endocrine- and chemo-therapy).     

Analytical partitioning of poly(ethylene glycol)-modified proteins

Covalently grafting proteins with varying numbers (n) of poly(ethylene glycol) molecules (PEGs) often enhances their biomedical and industrial usefulness. Partition between the phases in aqueous polymer two-phase systems can be used to rapidly characterize polymer-protein conjugates in a manner related to various enhancements. The logarithm of the partition coefficient (K) approximates linearity over the range 0<n<x. However, x varies with the nature of the conjugate (e.g., protein molecular mass) and such data analysis does not facilitate the comparison of varied conjugates. The known behavior of surface localized PEGs suggests a better correlation should exist between log K and the weight fraction of polymer in PEG-protein conjugates. Data from four independent studies involving three proteins (granulocyte-macrophage colony stimulation factor, bovine serum albumin and immunoglobulin G) has been found to support this hypothesis. Although somewhat simplistic, ‘weight fraction’ based analysis of partition data appears robust enough to accommodate laboratory to laboratory variation in protein, polymer and phase system type. It also facilitates comparisons between partition data involving disparate polymer-protein conjugates.     

Squaric acid mediated synthesis and biological activity of a library of linear and hyperbranched poly(glycerol)-protein conjugates

Polymer-protein conjugates generated from side chain functional synthetic polymers are attractive because they can be easily further modified with, for example, labeling groups or targeting ligands. The residue specific modification of proteins with side chain functional synthetic polymers using the traditional coupling strategies may be compromised due to the nonorthogonality of the side-chain and chain-end functional groups of the synthetic polymer, which may lead to side reactions. This study explores the feasibility of the squaric acid diethyl ester mediated coupling as an amine selective, hydroxyl tolerant, and hydrolysis insensitive route for the preparation of side-chain functional, hydroxyl-containing, polymer-protein conjugates. The hydroxyl side chain functional polymers selected for this study are a library of amine end-functional, linear, midfunctional, hyperbranched, and linear-block-hyperbranched polyglycerol (PG) copolymers. These synthetic polymers have been used to prepare a diverse library of BSA and lysozyme polymer conjugates. In addition to exploring the scope and limitations of the squaric acid diethylester-mediated coupling strategy, the use of the library of polyglycerol copolymers also allows to systematically study the influence of molecular weight and architecture of the synthetic polymer on the biological activity of the protein. Comparison of the activity of PG-lysozyme conjugates generated from relatively low molecular weight PG copolymers did not reveal any obvious structure-activity relationships. Evaluation of the activity of conjugates composed of PG copolymers with molecular weights of 10000 or 20000 g/mol, however, indicated significantly higher activities of conjugates prepared from midfunctional synthetic polymers as compared to linear polymers of similar molecular weight.     

Exploration of Moraxella catarrhalis outer membrane proteins, CD and UspA, as new carriers for lipooligosaccharide-based conjugates

Moraxella catarrhalis outer membrane proteins, CD and ubiquitous surface protein A (UspA), were used as carriers for M. catarrhalis detoxified lipooligosaccharide (dLOS)-based conjugates. Our study was designed to investigate the feasibility of CD and UspA as protein carriers for dLOS-based conjugates and their possible synergic effects on protection from both anti-LOS and anti-CD or anti-UspA antibody responses. Female Balb/c mice were immunized subcutaneously three times with dLOS-CD or dLOS-UspA conjugate in Ribi adjuvant. Antisera elicited by the conjugates showed high titers of specific anti-LOS antibodies with complement-dependent bactericidal activity towards M. catarrhalis strain 25238. In a mouse aerosol challenge model, mice immunized with both conjugates showed a significant enhancement of the clearance of strain 25238 from lungs as compared with the control mice. Although both conjugates elicited reduced (relative to unconjugated CD or UspA) but significant levels of anti-CD or UspA antibodies, they did not show synergetic effects with anti-LOS antibodies on the bactericidal activity or the pulmonary bacterial clearance. Nevertheless, CD and UspA are safe and effective new carriers for dLOS-based or other potential carbohydrate-based conjugate vaccines to help thymus-independent carbohydrate antigens for production of anti-carbohydrate antibodies against target pathogens.     

Evaluation of homologous, heterologous, and affinity conjugates for the serodiagnosis of Toxoplasma gondii and Neospora caninum in maned wolves (Chrysocyon brachyurus)

Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful.     

Separation of PEGylated alpha-lactalbumin from unreacted precursors and byproducts using ultrafiltration

There is considerable clinical interest in the use of "second-generation" therapeutic proteins produced by conjugation of the native protein with various polymers including poly(ethylene glycol) (PEG). One of the challenges in the production of polymer-protein conjugates is the need to remove residual polymer, native (unreacted) protein, and any reaction byproducts from the final therapeutic formulation. The overall objective of this study was to evaluate the possibility of using ultrafiltration for the purification of a model PEGylated protein. Sieving data were obtained using PEGylated alpha-lactalbumin, the native protein, and the poly(ethylene glycol) over a range of pH, ionic strength, and filtrate flux using both neutral and charge-modified composite regenerated cellulose membranes. Purification of the PEGylated protein was achieved using a two-stage diafiltration process. The first stage used a neutral membrane to remove the unreacted protein and any small reaction byproducts while retaining the large PEGylated product. The second stage used a negatively charged membrane to remove the neutral poly(ethylene glycol) while retaining the PEGylated alpha-lactalbumin as a result of strong electrostatic interactions. These results clearly demonstrate the potential of using membrane-based separations for the purification of second-generation therapeutic proteins.     

Polymer--drug conjugates as nano-sized medicines

Polymer Therapeutics have enormously evolved in the past decades. Several polymeric drugs as well as polymer-protein conjugates have been in the market since the 90s, but although polymer-drug conjugates are already in clinical trials they still need to reach this final goal. There are four main convergent strategies to move this platform technology further. First, exploitation of new molecular targets in cancer therapy and design of polymer-drug conjugates as treatments for other diseases. Second, the development of combination therapy. Third, attempts to improve polymer chemistry, including the use of new well-defined architectures and the optimization of the advanced characterization techniques essential to transform a promising conjugate into a candidate for clinical evaluation. Finally, increased understanding of polymer conjugate features that govern clinical risk-benefit is leading to an appreciation of clinical biomarkers that will open new possibilities for personalized therapy.     

甜菜夜蛾触角结合蛋白Ⅱ的cDNA克隆、 组织分布及配体结合特性分析

触角结合蛋白（antennal binding proteins, ABPs）是气味结合蛋白（odorant binding proteins, OBPs）的一个亚类, 推测其在昆虫嗅觉中起作用。为了探讨这一问题, 本研究通过转录组数据分析并利用RACE技术, 克隆了甜菜夜蛾Spodoptera exigua触角结合蛋白Ⅱ基因（SexigABP2）的全长cDNA序列（GenBank登录号为HQ234486）。序列分析表明, 该基因开放阅读框长444 bp, 编码148个氨基酸, 具有OBPs典型的6个半胱氨酸位点; 其氨基酸序列和烟芽夜蛾Heliothis virescens的HvirABP2的一致性最高, 达72%。实时定量PCR分析显示, 该基因主要在触角中表达, 在喙、 足、 翅等组织中也有少量表达, 且在雌蛾触角及足中的表达量显著高于雄蛾。进一步对该基因进行原核表达和纯化, 利用荧光竞争结合实验测定了SexigABP2对35种气味物质的结合能力, 发现其对甜菜夜蛾性信息素组分(Z)-9-十四碳烯醇和植物挥发物法尼醇的结合能力较强, 结合常数分别为8.24 μmol/L和8.14 μmol/L。结合能力比较表明, SexigABP2对不饱和长碳链化合物较饱和短碳链化合物具有更强的结合能力; 在不饱和长碳链化合物中, 对醇类物质又较乙酸酯类物质具有更强的结合能力。结果提示SexigABP2可能参与了成虫对不饱和长碳链的植物挥发物的感受。     

Lingshuiol, a novel polyhydroxyl compound with strongly cytotoxic activity from the marine dinoflagellate Amphidinium sp

A novel polyhydroxy compound with a linear carbon-chain, lingshuiol (1), had been isolated from the cultured marine dinoflagellate Amphidinium sp. Its structure was elucidated by extensive analysis of 2D NMR spectral data. Lingshuiol possessed a powerful cytotoxic activity against A-549 and HL-60 cells in vitro with the IC(50) of 0.21 and 0.23 microM, respectively.     

Lipophilic conjugates of methotrexate with short-chain alkylamino acids as DHFR inhibitors. Synthesis, biological evaluation, and molecular modeling

Pursuing previous researches on lipophilic conjugates of methotrexate, aimed at over-crossing a form of transport resistance shown by some tumor cell lines toward the drug, a new series of derivatives is described in which the drug alpha- and gamma-carboxyl groups have been linked through amide bonds to short-chain alpha-alkylamino acids (4-6 carbon atoms). A specific NMR study was performed to delineate the stereochemistry of the conjugates. The inhibitory activity of these compounds against the target enzyme, (bovine liver) dihydrofolate reductase, and a sensitive (CCRF-CEM) and a transport-resistant tumor cell subline (CEM-MTX) were assessed. The conjugates showed the ability of retaining the same inhibitory activity also against the resistant cell subline, against which the parent drug was much less active than against the wild one; the alpha,gamma-bis(hexyl) derivative was the most active term of the series. Docking studies are in agreement with the proposed mode of interaction of these conjugates with the human DHFR.     

Single-drug multiligand conjugates: synthesis and preliminary cytotoxicity evaluation of a paclitaxel-dipeptide "scorpion" molecule

To improve the targeting properties of receptor-directed drug-peptide conjugates, a multiligand approach was proposed and a model "scorpion" conjugate (6, Figure 1), consisting of two peptide "claws" and a paclitaxel (PTX) "tail", was synthesized. The cell surface receptor-directed peptide used in this single-drug multiligand (SDML) model was a segment of the amphibian peptide bombesin (BBN) which had the Y6Q7W8A9V10G11H12L13M14-NH2 sequence, designated here as BBN[6-14] (2, Figure 2). Due to the lipophilic nature of both PTX and BBN[6-14], compound 6 had a low water solubility. To enhance the solubility, PEG derivatives of this conjugate were prepared with the polymer inserted either in the claws or in the tail regions. In a preliminary random screening, conjugate 6 showed superior cytotoxic activity in several GRPR-positive human cancer cell lines as compared to free PTX and two single-drug single-ligand (SDSL) conjugates. In a receptor blocking experiment, addition of excess unconjugated BBN[6-14] ligand reduced the cytotoxicity of conjugate 6, indicating the receptor-mediated mechanism of drug delivery. The PEG-derived conjugates showed activities which were intermediate between SDSL and the SDML congeners. Also, an increase in the number of the PEG segments lowered cytotoxicity, possibly due to steric hindrance against ligand-receptor binding. Taken together, these results demonstrate the potential of the multiligand approach in the design of receptor-targeting conjugates for tumor-specific drug delivery.     

Antiserum to lucifer yellow: preparation, characterization, and use for immunocytochemical localization of dye-filled retinal neurons

We present a new method for the preparation of antisera to Lucifer Yellow, and these antisera are here shown to be particularly suitable for immunocytochemical localization of multiple dye-injected cells in large pieces of vertebrate retina. The method involves the preparation of covalent conjugates of the VS isomer of Lucifer Yellow with keyhole limpet hemocyanin (KLH) or rabbit serum albumin (RSA), and their use as immunogens in rabbits. Both carrier protein conjugates yielded robust antibody responses. Antiserum to the KLH-LY conjugate contained precipitating antibodies against LY and KLH, although activity to the latter did not interfere with immunocytochemical staining. Rabbit antiserum to the RSA-LY conjugate contained precipitating antibody only against LY. When used for immunocytochemical staining of large retinal pieces containing many LY-filled cells, both antisera yielded well-stained, darkly filled cells similar to those seen with the Golgi technique; even very fine dendritic processes of retinal ganglion cells could be followed for long distances. LY immunocytochemistry provides a useful alternative to photooxidation for the analysis of multiple dye injected cells, especially in whole mounts. This approach may also be useful for immunocytochemical identification of cells filled with LY after tissue fixation.     

肺炎球菌表面蛋白A(PspA)及其荚膜多糖交联物的免疫原性和交叉保护作用

分别采用肺炎球菌表面蛋白A(PspA)和PspA-荚膜多糖交联物免疫小鼠,研究PspA及其交联物的免疫特性.用酶联免疫吸附法(ELISA)检测抗原的免疫原性,用动物保护试验检验抗原对肺炎球菌6B,5,1,23F,19F型的交叉免疫效果.实验结果表明:肺炎球菌表面蛋白A及其多糖交联物表现出一定的交叉保护作用,具有较好的应用前景.     

Role of the methoxy group in immune responses to mPEG-protein conjugates

Anti-PEG antibodies have been reported to mediate the accelerated clearance of PEG-conjugated proteins and liposomes, all of which contain methoxyPEG (mPEG). The goal of this research was to assess the role of the methoxy group in the immune responses to mPEG conjugates and the potential advantages of replacing mPEG with hydroxyPEG (HO-PEG). Rabbits were immunized with mPEG, HO-PEG, or t-butoxyPEG (t-BuO-PEG) conjugates of human serum albumin, human interferon-α, or porcine uricase as adjuvant emulsions. Assay plates for enzyme-linked immunosorbent assays (ELISAs) were coated with mPEG, HO-PEG, or t-BuO-PEG conjugates of the non-cross-reacting protein, porcine superoxide dismutase (SOD). In sera from rabbits immunized with HO-PEG conjugates of interferon-α or uricase, the ratio of titers of anti-PEG antibodies detected on mPEG-SOD over HO-PEG-SOD ("relative titer") had a median of 1.1 (range 0.9-1.5). In contrast, sera from rabbits immunized with mPEG conjugates of three proteins had relative titers with a median of 3.0 (range 1.1-20). Analyses of sera from rabbits immunized with t-BuO-PEG-albumin showed that t-butoxy groups are more immunogenic than methoxy groups. Adding Tween 20 or Tween 80 to buffers used to wash the assay plates, as is often done in ELISAs, greatly reduced the sensitivity of detection of anti-PEG antibodies. Competitive ELISAs revealed that the affinities of antibodies raised against mPEG-uricase were c. 70 times higher for 10 kDa mPEG than for 10 kDa PEG diol and that anti-PEG antibodies raised against mPEG conjugates of three proteins had >1000 times higher affinities for albumin conjugates with c. 20 mPEGs than for analogous HO-PEG-albumin conjugates. Overall, these results are consistent with the hypothesis that antibodies with high affinity for methoxy groups contribute to the loss of efficacy of mPEG conjugates, especially if multiply-PEGylated. Using monofunctionally activated HO-PEG instead of mPEG in preparing conjugates for clinical use might decrease this undesirable effect.     

Effect of conjugation methodology on the immunogenicity and protective efficacy of meningococcal group C polysaccharide-P64k protein conjugates

Neisseria meningitidis serogroup C polysaccharide (CCPS) was conjugated to the carrier protein P64k using two different conjugation procedures, condensation mediated by carbodiimide with adipic acid dihydrazide as spacer and the reductive amination method. BALB/c mice were immunized with the resultant polysaccharide-protein conjugates and the immune response was evaluated. All conjugates assayed generated at least 10-fold higher antibody titers than the free polysaccharide. The reductive amination method rendered the best conjugate (CCPS-P64kR) that was able to elicit antibody titers statistically higher than the titer elicited by the plain CCPS (P<0.001). The sera of the group immunized with CCPS-P64kR showed a three-fold higher bactericidal response than the sera of the group immunized with the plain CCPS and they were able to protect against challenge with meningococci in the infant rat protection model. In addition, three different conjugates were obtained from polysaccharides with molecular relative sizes of 2000-4000 Da, 4000-10,000 Da or 10,000-50,000 Da, but no differences were detected in the immune response obtained against the three conjugates. Our experiments demonstrate that it is possible to generate a protective, T-cell-dependent response against CCPS using the P64k protein as carrier.     

Preparation of specific antisera to estrone-3-glucuronide and estriol-3-glucuronide

The preparation and antigenic properties of estrone-3-glucuronide- and estriol-3-glucuronide-bovine serum albumin conjugates in which the hapten is linked to the carrier protein through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Antibodies raised against the two immunogens in the rabbit possessed high specificity to estrone-3-glucuronide and estriol-3-glucuronide, respectively, exhibiting little cross-reactivities with other estrogen conjugates and no cross-reactions with related steroids except for free estrogens, their 3-methyl ethers and 3-sulfates. The cross-reactive antibodies were eliminated by partial immunoadsorption on affinity chromatographic media using the estrone-3-methyl ether 17-(O-carboxymethyl)oxime- and estriol-3-methyl ether 16 (or 17)-hemisuccinate-aminohexyl Sepharose conjugates, respectively. The purified antisera exhibited no cross-reactivities with free estrogens and ring A conjugates of estrone and estriol.