Comparative studies of irinotecan-loaded polyethylene glycol-modified liposomes prepared using different PEG-modification methods

Recently, a polyethylene glycol (PEG)-modification method for liposomes prepared using pH-gradient method has been proposed. The differences in the pharmacokinetics and the impact on the antitumor effect were examined; however the impact of PEG-lipid molar weight has not been investigated yet. The main purpose of this study is to evaluate the impact of PEG-lipid molar weight against the differences in the pharmacokinetics, the drug-release profile, and the antitumor effect between the proposed PEG-modification method, called the post-modification method, and the conventional PEG-modification method, called the pre-modification method. Various comparative studies were performed using irinotecan as a general model drug. The results showed that PEG-lipid degradation could be markedly inhibited in the post-modification method. Furthermore, prolonged circulation time was observed in the post-modification method. The sustained drug-release was observed in the post-modification method by the results of the drug-releasing test in plasma. Moreover, a higher antitumor effect was observed in the post-modification method. It was also confirmed that the same behaviors were observed in all comparative studies even though the PEG molecular weight was lower. In conclusion, the post-modification method has the potential to be a valuable PEG-modification method that can achieve higher preservation stability of PEG-lipid, prolonged circulation time, and higher antitumor effect with only half the amount of PEG-lipid as compared to the pre-modification method. Furthermore, it was demonstrated that PEG(5000)-lipid would be more desirable than PEG(2000)-lipid since it requires much smaller amount of PEG-lipid to demonstrate the same performances.     

Stabilized plasmid-lipid particles containing PEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression

Stabilized plasmid lipid particles (SPLP) consist of a single copy of DNA surrounded by a lipid bilayer. The particles are small ( approximately 100 nm), stable, monodisperse and have a low surface charge. A diffusible polyethylene glycol (PEG) coating attached to a lipid anchor is critical to the SPLP's functionality. The PEG-lipid exchanges out of the bilayer at a rate determined by the size of the lipid anchor. Here we show that SPLP can be prepared using a series of PEG-diacylglycerol lipids (PEG-S-DAGs). SPLP were prepared incorporating PEG-dimyristoylglycerol (C14), PEG-dipalmitoylglycerol (C16) or PEG-distearoylglycerol (C18) and the rate of PEG-lipid diffusion from the bi-layer determined using a FRET assay. SPLP pharmacokinetics confirm a correlation between the stability of the PEG-lipid component and circulation lifetime. PEG-S-DAGs with longer lipid anchors yield more stable SPLP particles with longer circulation half-lives yielding an increase in tumor delivery and gene expression. PEG-distearoylglycerol (C18) containing SPLP bypass so-called 'first pass' organs, including the lung, and elicit levels of gene expression in distal tumor tissue 100- to 1000-fold greater than that observed in any other tissue. The incorporation of PEG-S-DAG in SPLP confirms that small size, low surface charge and extended circulation lifetimes are prerequisite to the accumulation and tumor selective expression of plasmid DNA following systemic administration.     

Identification of the modifying sites of mono-PEGylated salmon calcitonins by capillary electrophoresis and MALDI–TOF mass spectrometry

A capillary electrophoretic method (CE) was developed for the determination of the PEG-modification sites of three positional isomers of mono-PEG modified salmon calcitonins (mono-PEG-sCTs). Resistance to proteolytic degradation on the PEG modification sites resulted in different patterns of CE electropherograms for the tryptic digested mono-PEG-sCTs isomers, and the PEG modification sites were assigned accordingly. The PEG-modification sites were also confirmed directly by determining the molecular masses of the tryptic digested PEG-modified fragments of respective mono-PEG-sCT by the matrix-assisted laser desorption ionization time-of-flight (MALDI–TOF) mass spectrometry.     

Evaluation of Spray Cycle Bioreactor Using L-Alanine Degradation by Candida maltosa

The oxygen-supply capability of a spray cycle reactor was evaluated by using it for oxidative degradation of L-alanine. The volumetric oxygen transfer coefficient, k_La, was evaluated as a parameter for the oxygen supply. The liquid circulation rate in the spray cycle reactor was represented in terms of the number of circulations. The k_La increased with the number of circulations, especially by stirring in the reservoir vessel, reaching 272/h at 4.4/min of circulation numbers. This value was 1.4 times higher than that without stirring. The L-alanine degradation rate increased as the cell growth was promoted, and as the circulation numbers increased. Finally, the spray cycle reactor was evaluated by the specific degradation rate. This rate increased in proportion to the k_La, and was 8.8 times higher than that in the jar fermentor, suggesting that the spray cycle reactor is superior for oxygen-demanding fermentation.     

Stabilized plasmid-lipid particles containing PEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression

Stabilized plasmid lipid particles (SPLP) consist of a single copy of DNA surrounded by a lipid bilayer. The particles are small (∼100 nm), stable, monodisperse and have a low surface charge. A diffusible polyethylene glycol (PEG) coating attached to a lipid anchor is critical to the SPLP's functionality. The PEG-lipid exchanges out of the bilayer at a rate determined by the size of the lipid anchor. Here we show that SPLP can be prepared using a series of PEG-diacylglycerol lipids (PEG-S-DAGs). SPLP were prepared incorporating PEG-dimyristoylglycerol (C₁₄), PEG-dipalmitoylglycerol (C₁₆) or PEG-distearoylglycerol (C₁₈) and the rate of PEG-lipid diffusion from the bi-layer determined using a FRET assay. SPLP pharmacokinetics confirm a correlation between the stability of the PEG-lipid component and circulation lifetime. PEG-S-DAGs with longer lipid anchors yield more stable SPLP particles with longer circulation half-lives yielding an increase in tumor delivery and gene expression. PEG-distearoylglycerol (C₁₈) containing SPLP bypass so-called ‘first pass’ organs, including the lung, and elicit levels of gene expression in distal tumor tissue 100- to 1000-fold greater than that observed in any other tissue. The incorporation of PEG-S-DAG in SPLP confirms that small size, low surface charge and extended circulation lifetimes are prerequisite to the accumulation and tumor selective expression of plasmid DNA following systemic administration.     

Reversal of P-glycoprotein-mediated multidrug resistance by doxorubicin and quinine co-loaded liposomes in tumor cells

Multidrug resistance (MDR) is a major obstacle to successful clinical cancer chemotherapy. Currently, there is still unsatisfactory demand for innovative strategies as well as effective and safe reversing agent to overcome MDR. In this study, we developed a novel nanoformulation, in which doxorubicin hydrochloride (DOX) and quinine hydrochloride (QN) were simultaneously loaded into liposomes by a pH-gradient method for overcoming MDR and enhancing cytotoxicity in a doxorubicin-resistant human breast cancer cell line (MCF-7/ADR). The various factors were investigated to optimize the formulation and manufacturing conditions of DOX and QN co-loaded liposomes (DQLs). The DQL showed uniform size distribution and high encapsulation efficiency (over 90%) for both the drugs. Furthermore, DQLs significantly displayed high intracellular accumulation and potential of MDR reversal capability in MCF-7/ADR cells through the cooperation of DOX with QN, in which QN played the role as a MDR reversing agent. The IC₅₀ of DQL0.5:1 with the DOX/QN/SPC weight ratio of 0.5:1:50 was 1.80?±?0.03?μg/mL, which was 14.23 times lower than that of free DOX in MCF-7/ADR cells. And the apoptotic percentage induced by DQL0.5:1 was also increased to 62.2%. These findings suggest that DQLs have great potential for effective treatment of MDR cancer.     

Computational Prediction of Human Salivary Proteins from Blood Circulation and Application to Diagnostic Biomarker Identification

Proteins can move from blood circulation into salivary glands through active transportation, passive diffusion or ultrafiltration, some of which are then released into saliva and hence can potentially serve as biomarkers for diseases if accurately identified. We present a novel computational method for predicting salivary proteins that come from circulation. The basis for the prediction is a set of physiochemical and sequence features we found to be discerning between human proteins known to be movable from circulation to saliva and proteins deemed to be not in saliva. A classifier was trained based on these features using a support-vector machine to predict protein secretion into saliva. The classifier achieved 88.56% average recall and 90.76% average precision in 10-fold cross-validation on the training data, indicating that the selected features are informative. Considering the possibility that our negative training data may not be highly reliable (i.e., proteins predicted to be not in saliva), we have also trained a ranking method, aiming to rank the known salivary proteins from circulation as the highest among the proteins in the general background, based on the same features. This prediction capability can be used to predict potential biomarker proteins for specific human diseases when coupled with the information of differentially expressed proteins in diseased versus healthy control tissues and a prediction capability for blood-secretory proteins. Using such integrated information, we predicted 31 candidate biomarker proteins in saliva for breast cancer.     

Photoinitiated destabilization of sterically stabilized liposomes

A considerable effort has been devoted to the development of liposomes for the transport and buffering of drugs in the body. Several research groups have reported the increased localization of sterically stabilized liposomes (PEG-liposomes) at tumor sites. If PEG-liposomes are to be effective carriers of therapeutic agents, their drug permeability must be sufficiently low that little passive release occurs during the circulation time of the PEG-liposomes. However, once PEG-liposomes reach tumor sites, it may be desirable to accelerate the release of the encapsulated drug. The use of light to stimulate the release of encapsulated compounds from liposomes is attractive, because it is possible to control the spatial and temporal delivery of the radiation. PEG-liposomes composed in part of the photosensitive lipid, bis-SorbPC, can be prepared in a manner that effectively encapsulates water soluble compounds, yet releases them upon exposure to ultraviolet light in the presence of oxygen. The observed increase in liposome permeability is about 200-fold at high photoconversion of the monomeric bis-SorbPC. The increase in permeability is dependent on the extent of photolysis, but independent of both the charge on the PEG-lipid and the mole fraction of PEG-lipid included in the liposome. Therefore the photoinitiated destabilization of these PEG-liposomes is not a consequence of micellization of the PEG-lipid, but probably due to the formation of defects in the bilayer during crosslinking of the bis-SorbPC. The photoinduced increase in liposome permeability is great enough to make it possible to release therapeutic agents from PEG-liposomes at specific sites in a manner of tens of minutes to hours.     

Quantitation of soluble aggregates in recombinant monoclonal antibody cell culture by pH-gradient protein A chromatography

Monoclonal antibodies (mAbs) produced from mammalian cell culture may contain significant amounts of dimers and higher order aggregates. Quantitation of soluble aggregates in the cell culture is time-consuming and labor-intensive, usually involving a purification step to remove the impurities that interfere with the subsequent size exclusion chromatography (SEC) analysis. We have developed a novel pH-gradient protein A chromatography for rapid, non-size based separation of the aggregates in mAb cell culture samples. Our results demonstrate that this method has excellent correlation with SEC and can be applied to both human immunoglobulin gamma 1 (IgG1) and IgG2 antibodies. This approach can be useful in the quantitation of soluble aggregates in crude cell culture samples.     

Doxorubicin and chloroquine coencapsulated liposomes: preparation and improved cytotoxicity on human breast cancer cells

Doxorubicin, as a widely used chemotherapeutic, always causes multidrug resistance in human cancer cells. To circumvent drug resistance, we developed a novel formulation where doxorubicin hydrochloride (DOX) and chloroquine phosphate (CQ) were simultaneously loaded into liposomes by a pH-gradient method where CQ played the role of a chemical sensitizer. The various factors were investigated to optimize the formulation and manufacturing conditions of DOX and CQ coencapsulated liposomes (DCL). The resultant DCLs achieved the high encapsulation efficiency of both drugs over 90%. Further, DCLs significantly displayed resistance reversal action on a doxorubicin-resistant human breast cancer cell line (MCF-7/ADR) through the cooperation of CQ with DOX. The reversal fold of DCL with the DOX/CQ/soybean phosphatidylcholine weight ratio of 0.5:1:50 was 5.7, compared to free DOX. These results demonstrate that DCL is a promising formulation for the treatment of DOX-resistant breast cancer.     

Localized real time blood flow measurements

A novel method for real time, localized, flow measurements is applied to blood flow in human fingers. Results for arterial and venous flow in normal subjects and patients with abnormal blood circulation are presented. Effects of blood flow regulation by the autonomic nervous system have been observed. Stricture of the digital arteries could be clearly demonstrated in a patient with Raynaud's phenomenon. Experimental signals due to pulsatile flow in a model system can be simulated in a quantitative way. The calibration, however, depends on the actual spin-spin relaxation time and the shape of the pulsatile flow vs. time curve. Due to these limitations, the volume flow rate can be measured with a relative error of approximately +/- 25%.     

Do electrostatic interactions determine glycation of hyaluronidase derivatives with N-acetylhexosamines?

Using N-acetylglucosamine and N-acetylgalactosamine as model agents for glycation of native hyaluronidase and its chondroitin sulfate modified form it has been shown that the modified enzyme exhibited higher inactivation than the native enzyme, while heparin caused similar inhibition of both forms. Such effect could be attributed to the development of electrostatic interactions as the modified hyaluronidase had altered surface electrostatic potential after chondroitin sulfate binding. However, variations in ionic strength of the medium containing enzyme derivatives have shown that their endoglycosidase activity changed in a similar manner and the effect on glycation represents a multifactor process. N-acetylhexosamines are natural labels of endothelial glycocalyx degradation products. Interaction of the hyaluronidase forms with charged hyaluronan fragments revealed significantly higher inactivation of the modified enzyme compared with the native enzyme. The glycation pattern observed in this study was opposite to that observed with mono- and disaccharides. Thus, it appears that the investigated hyaluronidase derivatives represent an informative enzymatic test in vivo for determination of the dominant type of glycation agents in blood circulation and their origin.     

Impacts of hydrodynamic conditions on sludge digestion in internal circulation anaerobic digester

An investigation of hydrodynamic conditions and their impacts on sludge digestion in internal circulation anaerobic digester (ICAD) was conducted. This novel sludge digester employing upflow reactor concept was composed of reaction zone, riser and downcomer, etc. Part of the generated biogas was injected to the reaction zone to intensify the circulation in ICAD in order to enhance the mass transfer. The advanced particle image velocimetry was used to explore the hydrodynamic conditions at micro scale. The results revealed that the average shear rate in the reaction zone and downcomer was linearly correlated with the Reynolds number; the shear rates in ICAD revealed an order as riser > downcomer > reaction zone. Thermophilic digestion of waste activated sludge in a pilot ICAD for 5.25 d combined with thermal pretreatment at 60 °C for 1 d was conducted under various hydrodynamic conditions. The survey at global level demonstrated that both the longer sludge retention time than the hydraulic retention time and the satisfactory mass transfer could be realized and as consequences, the maximal biogas production rate and VSS removal were obtained when the Reynolds number of the reaction zone was approximately 0.53. This digester under the optimal hydrodynamic conditions demonstrated good degradation capacity and buffer capability to resist various shock loadings.     

几种耐热戴氏霉对秸秆的降解效果

【目的】测定6株戴氏霉(Taifanglania)的生长温度特性及其对秸秆的降解效果。【方法】通过定时测定不同培养温度下的菌落直径绘制6株戴氏霉菌株的生长曲线;采用苯胺蓝法、愈创木酚法和木质素磺酸钙降解试验测定其木质素降解能力;用羧甲基纤维素钠水解圈测定法和胞外酶活测定法判定其对纤维素的降解能力;以失重法和范氏洗涤剂法检测其对水稻秸秆的降解效果。【结果】所试的耐热戴氏霉菌株均能耐受50 °C的高温,并能产生纤维素酶,但不同菌株产生的木质素降解酶有所差异;均具有降解秸秆的能力,其中合川戴氏霉(T. hechuanensis) H08.1菌株降解能力最强,其次是灰戴氏霉(T. cinerea) H57.1菌株,其秸秆降解率分别为50.2%和42.2%。【结论】合川戴氏霉H08.1菌株和灰戴氏霉H57.1菌株在秸秆的降解利用上具有潜在开发价值。     

Proliferation characteristics and polyploidization of cultured myofibroblasts from a patient with fibroblastic rheumatism

Fibroblast-like cells were obtained from a nodule of a patient with fibroblastic rheumatism, and grown in culture for different times (from passage 3 to 21). These cells as well as the fibroblasts taken from an unaffected skin area (controls) of the same patient, have been investigated by fluorescence microscopy, cytochemical methods and cytometry, to evaluate their cytodifferentiation features and cytokinetic characteristics. In addition, in low-passage cultures, the secretion of collagen and of non-collagenic proteins was evaluated using electrophoretic techniques. The immunolabeling with antibodies against sm-specific a-actin (which was taken as a marker of myofibroblasts) showed that, already in low-passage cultures, the percentage of myofibroblasts was higher in the nodule-derived cell populations, and progressively increased with increasing passages. This suggests that myofibroblasts have higher proliferation potential than control fibroblasts. Myofibroblasts were also found to undergo polyploidization and hypertrophy, especially in high-passage cultures. Based on these results, it may be hypothesized that in fibroblastic rheumatism the development of the typical nodules could depend on the intrinsic capability of myofibroblats of proliferating faster than normal fibroblasts and of becoming polyploid and hypertrophic. Nodule-derived cells in culture synthesized slightly less collagen and non-collagen proteins than did the control fibroblasts; this suggests that the increased fibrosis observed in nodules in situ could be likely dependent on a reduced degradation of the extracellular matrix components.     

植物萜类生物合成中的后修饰酶

萜类化合物由于其结构类型丰富多样而被称为"terpenome".除了参与植物生长发育、环境应答等生理过程,萜类化合物还应用于医药、有机化工等领域.萜类的生物合成大致可分为前体形成、骨架构建以及后修饰三部分,基本骨架通常由萜类合酶催化形成,进一步在后修饰酶的作用下产生数以万计的萜类化合物.结合我们对香茶菜二萜生物合成的初步研究结果,本文主要针对近年来植物萜类生物合成中的一些有代表性的后修饰酶包括P450单氧酶、双键还原酶、酰基转移酶和糖基转移酶,进行研究现状分析与展望.     

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.     

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.     

Effects of PEG-lipids on permeability of phosphatidylcholine/cholesterol liposomes in buffer and in human serum

The permeability of liposomal membranes was studied as a function of the amount of incorporated PEG-lipid. The fluorescent dyes ethidium, propidium and 5(6)-carboxy fluorescein were used as markers for measurements of spontaneous leakage. The results show that addition of up to 8 mol% of PEG(2000)-DSPE into liposomal membranes of DSPC/Cho and EPC/Cho reduces the permeability of carboxyfluorescein in buffer solution. In contrast, the leakage of the more amphiphilic dye ethidium was not to any measurable extent affected by PEG-lipid inclusion. Another important difference was that ethidum leakage showed a clear dependence on temperature whereas leakage of carboxyfluorescein from pegylated liposomes did not. We conclude that the mechanisms by which the two dyes permeate the liposomal bilayer are qualitatively different. Both ethidium and carboxyfluorescein did interact with human serum components in a way that made measurements in serum unreliable. The more hydrophilic ethidium analogue propidium was shown not to interact with human serum components to any detectable extent. This made propidium suitable for permeability determinations in human serum. It was found that liposomes composed of pure EPC or EPC with 5 mol% DSPE-PEG, displayed a dramatic increase in permeability when subjected to a medium composed of 20% human serum in buffer. Addition of 40 mol% cholesterol to the EPC bilayers reduced the observed release rate in human serum substantially, whereas no stabilizing effect was observed upon PEG-lipid inclusion.     

Preparation and Characterization of Stable pH-Sensitive Vesicles Composed of α-Tocopherol Hemisuccinate

The current study aims to develop a stable pH-sensitive drug delivery system. First, cleavable polyethylene glycol-α-tocopherol hemisuccinate (PEG-THS) was synthesized. Conventional pH-sensitive vesicles composed of the Tris salt of α-tocopherol hemisuccinate (THST) were then prepared using the detergent removal technique. The vesicles had a mean particle size of (163.8 ± 5.5) nm and a zeta potential of −74.5 ± 6.4 mV. The THST vesicles were then modified using PEG-THS or uncleavable PEG-cholesterol (PEG-CHOL) (THST/PEG-lipids, 100:6 molar ratio). The mean vesicle particle size and absolute zeta potential decreased with increasing PEG-THS proportion. When the pH was decreased, the vesicle particle size and calcein release rate increased. The THST vesicles were initially Ca²⁺-unstable but exhibited significantly improved stability after modification with PEG-THS, especially at PEG-lipid ratios above 6%. Incubation in an acid serum increased the calcein release rate of conventional THST vesicles to 45 ± 1.98% at 10 min. However, the release rate of the PEG-CHOL vesicles remained low. The calcein release rate of PEG-THS vesicles was between those of conventional and PEG-CHOL-V. Therefore, PEG-THS can protect vesicles in serum and reconstitute their pH sensitivity in acidic conditions. Cleavable PEG-THS can be used in stable pH-sensitive preparations without loss of pH sensitivity. Free calcein and conventional vesicles eliminated from the plasma soon after injection, as well as the half-life (t_1/2) and area under the curve of PEG-THS-V encapsulating calcein, were dramatically increased. This phenomenon indicates that the use of PEG-lipid derivatives has gained a favorably long circulation effect in mice.Key words: cleavage, long circulation, PEG-α-tocopherol hemisuccinate, pH-sensitive, vesicles