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Developing efficient catalysts to produce clean fuel by using solar energy has long been the goal to mitigate the issue of traditional fossil fuel scarcity. In this work, we design a heterostructure photocatalyst by employing two green components, Ni(OH)

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.PEG 修饰被认为是改善重组蛋白药物特性的最有效手段，包括增加蛋白质药物在体内的血浆半衰期，降低免疫原性和抗原性。目前典型的PEG 修饰手段为将PEG 连接至蛋白质的游离氨基，包括赖氨酸和N-末端，但这种连接缺乏选择性，产物为混合物，活性及工艺稳定性差，难以控制。酶法PEG 化修饰能有效克服上述缺点，其中谷氨酰胺转氨酶 (TGase) 可以作为PEG 化定点修饰用酶。文中选择重组人干扰素α2a (IFN α2a) 进行酶法修饰反应，通过计算机模拟预测IFN α2a 可以在第101 位Gln 特异性定点修饰。将IFN α2a 与40 kDa 的Y 型PEG 在微生物来源的谷氨酰胺转氨酶 (mTG) 催化下进行定点PEG 化修饰。结果显示，mTG 可以介导IFN α2a 特异性位点Gln 的单一定点PEG 修饰，产生分子量为58 495.6 Da的PEG-Gln101-IFN α2a 分子。圆二色谱结果显示，PEG-Gln101-IFN α2a 与未修饰的IFN α2a 具有相同的二级结构。SD 大鼠药代结果显示，与IFN α2a 相比，PEG-Gln101-IFN α2a 能有效提高药代动力学参数，强于已上市PEGIFN α2a-PEGASYS®。.

β-Amyloid (Aβ) elevation, tau hyperphosphorylation, and neuroinflammation are major hallmarks of Alzheimer's disease (AD). Glycogen synthase kinase-3β (GSK-3β) is a key protein kinase implicated in the pathogenesis of AD. Blockade of GSK-3β is an attractive therapeutic strategy for AD. Isoorientin, a 6-C-glycosylflavone, was previously shown to be a highly selective inhibitor of GSK-3β, while exerting neuroprotective effects in neuronal models of AD. In the present study, we evaluated the in vivo effects of isoorientin on GSK-3β, tau phosphorylation, Aβ deposition, neuroinflammatory response, long-term potentiation, and spatial memory in amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice using biochemical, electrophysiological, and behavioral tests. Chronic oral administration of isoorientin to APP/PS1 mice at 8 months of age attenuated multiple AD pathogenic hallmarks in the brains, including GSK-3β overactivation, tau hyperphosphorylation, Aβ deposition, and neuroinflammation. For neuroinflammation, isoorientin treatment reduced the number of activated microglia associated with Aβ-positive plaques, and in parallel reduced the levels of pro-inflammatory factors in the brains of APP/PS1 mice. Strikingly, isoorientin reversed deficits in synaptic long-term potentiation and spatial memory relevant to cognitive functions. Together, the findings suggest that isoorientin is a brain neuroprotector and may be a promising drug lead for treatment of AD and related neurodegenerative disorders.

Isoorientin has anti-inflammatory effects; however, the mechanism remains unclear. We previously found isoorientin is an inhibitor of glycogen synthase kinase 3β (GSK3β) in vitro. Overactivation of GSK3β is associated with inflammatory responses. GSK3β is inactivated by phosphorylation at Ser9 (i.e., p-GSK3β). Lithium chloride (LiCl) inhibits GSK3β and also increases p-GSK3β (Ser9). The present study investigated the anti-inflammatory effect and mechanism of isoorientin via GSK3β regulation in lipopolysaccharide- (LPS-) induced RAW264.7 murine macrophage-like cells and endotoxemia mice. LiCl was used as a control. While AKT phosphorylates GSK3β, MK-2206, a selective AKT inhibitor, was used to activate GSK3β via AKT inhibition (i.e., not phosphorylate GSK3β at Ser9). The proinflammatory cytokines TNF-α, IL-6, and IL-1β were detected by ELISA or quantitative real-time PCR, while COX-2 by Western blotting. The p-GSK3β and GSK3β downstream signal molecules, including NF-κB, ERK, Nrf2, and HO-1, as well as the tight junction proteins ZO-1 and occludin were measured by Western blotting. The results showed that isoorientin decreased the production of TNF-α, IL-6, and IL-1β and increased the expression of p-GSK3β in vitro and in vivo, similar to LiCl. Coadministration of isoorientin and LiCl showed antagonistic effects. Isoorientin decreased the expression of COX-2, inhibited the activation of ERK and NF-κB, and increased the activation of Nrf2/HO-1 in LPS-induced RAW264.7 cells. Isoorientin increased the expressions of occludin and ZO-1 in the brain of endotoxemia mice. In summary, isoorientin can inhibit GSK3β by increasing p-GSK3β and regulate the downstream signal molecules to inhibit inflammation and protect the integrity of the blood-brain barrier and the homeostasis in the brain.

Mitoxantrone (MIT) was encapsulated into 60, 80 and 100nm pegylated hydrogenated soy phosphatidylcholine/cholesterol (HSPC/chol) vesicles using a transmembrane (NH(4))(2)SO(4) gradient. In-vitro release studies revealed that small-sized formulation had fast drug-release rate. Acute toxicity studies performed in c57 mice proved that all pegylated liposomal MIT (plm) formulations could be well-tolerated at a dose of 9mg/kg, significantly compared to severe toxicity induced by free mitoxantrone (f-M). In KM mice, plm60 was at least 2- to 3-fold less toxic than f-M. After intravenous injection, plm60 was slowly eliminated from plasma relative to f-M, resulting in about 6459-fold increase in AUC and its plasma kinetics exhibited dose dependence. In S-180 bearing KM mice, plm60 preferentially accumulated into tumor zone, with a approximately 12-fold increase in AUC and approximately 10-fold increase in C(max) Furthermore, the accumulation of plm60 in almost all normal tissues markedly decreased. The antitumor efficacy of plm60 was also considerably enhanced. In L1210 ascitic tumor model, plm60 was the most efficacious which led to a approximately 70% long-term survival, significantly compared to 16-33% survival rate in plm80, plm100 and f-M groups at the same dose level (4mg/kg). The antitumor efficacy of plm60 was more encouraging in L1210 liver metastasis model. At a dose of 6mg/kg, approximately 90% animals receiving plm60 treatment could survive 60 days; however, in f-M group at the same dose, all the mice died at approximately 14 days post inoculation. Similarly, plm60 could effectively inhibit the growth of RM-1 tumor in BDF1 mice, resulting in marked increase in tumor doubling time at different dose levels relative to f-M. The improved antineoplastic effects could be ascribed to its small vesicle size, which allowed more drug release after the accumulation into tumor zone. Theoretical considerations revealed that the reduction of vesicle size could increase the specific area of MIT/sulfate precipitate inside the vesicle and the release constant K, which is inversely proportional to vesicle volume (K=pA(m)k(2)k(2)(')/([H(+)](i)(2)V(i))).

Besides pH gradient, other transmembrane gradients such as metal ion gradient could be also employed to load drugs into liposomes. In pH gradient method, anions have an important role since they could form specific aggregates with drugs, and then affect drug release kinetics from vesicles. To explore the role of anions in metal ion gradient method, copper ion-mediated mitoxantrone (MIT) loading was investigated systematically. When empty liposomes exhibiting a transmembrane copper ion gradient (300 mM) were mixed with MIT in a molar ratio of 0.2:1, after 5 min incubation at 60 degrees C, >95% MIT could be loaded into vesicles and the encapsulation was stable, regardless of the kinds of anions and initial intraliposomal pH values. The encapsulation ratio decreased with increased MIT/lipid molar ratio. But even when the molar ratio increased to 0.4, >90% encapsulation could still be achieved. In the presence of nigericin and ammonium, the drug loading profiles were affected to different degree with respect to both drug loading rate and encapsulation ratio. Relative to CuSO(4)-containing systems, CuCl(2) mediated MIT loading was unstable. Both nigericin and ammonium could alter the absorption spectra of liposomal MITs loaded with CuSO(4) gradient. In vitro release studies were performed in glucose/histidine buffer and in 50% human plasma using a dialysis method. In both of release media, CuCl(2)-containing vesicles displayed rapid release kinetics in comparison with CuSO(4) systems; and during the experiment period, MIT was lost from the vesicles continuously. When the formulations were injected into BDF1 mice at a dose of 4 mg/kg, all the liposomal formulations exhibited enhanced blood circulation time, with half-life values of 6.8-7.2h, significantly compared to the rapid clearance of free-MIT. In L1210 ascitic model, CuCl(2) formulation was more therapeutically active than CuSO(4) formulation. At a dose of 6 mg/kg, the treatment with CuCl(2) formulation resulted in a median survival time of 21 days, considerably larger than that of CuSO(4) groups (15 days). Based on these data, it was concluded that during the drug loading process, a dynamic transmembrane pH gradient is generated and intraliposomal pH might affect the complexation manner in which Cu(2+) binds MIT. Owing to the presence of pH gradient, after the accumulation within vesicles, a part of MIT will be protonated and precipitated by sulfate. Accordingly, the aggregation status of MIT inside CuSO(4) system was more complicated than that in CuCl(2) vesicles. The difference in physical status of MIT aggregates affects not only the drug release rate, but also their therapeutic effects.