Bioinspired PROTAC-induced macrophage fate determination alleviates atherosclerosis.

Atherosclerosis is a major cause of death and disability in cardiovascular disease. Atherosclerosis associated with lipid accumulation and chronic inflammation leads to plaques formation in arterial walls and luminal stenosis in carotid arteries. Current approaches such as surgery or treatment with statins encounter big challenges in curing atherosclerosis plaque. The infiltration of proinflammatory M1 macrophages plays an essential role in the occurrence and development of atherosclerosis plaque. A recent study shows that TRIM24, an E3 ubiquitin ligase of a Trim family protein, acts as a valve to inhibit the polarization of anti-inflammatory M2 macrophages, and elimination of TRIM24 opens an avenue to achieve the M2 polarization. Proteolysis-targeting chimera (PROTAC) technology has emerged as a novel tool for the selective degradation of targeting proteins. But the low bioavailability and cell specificity of PROTAC reagents hinder their applications in treating atherosclerosis plaque. In this study we constructed a type of bioinspired PROTAC by coating the PROTAC degrader (dTRIM24)-loaded PLGA nanoparticles with M2 macrophage membrane (MELT) for atherosclerosis treatment. MELT was characterized by morphology, size, and stability. MELT displayed enhanced specificity to M1 macrophages as well as acidic-responsive release of dTRIM24. After intravenous administration, MELT showed significantly improved accumulation in atherosclerotic plaque of high fat and high cholesterol diet-fed atherosclerotic (ApoE-/-) mice through binding to M1 macrophages and inducing effective and precise TRIM24 degradation, thus resulting in the polarization of M2 macrophages, which led to great reduction of plaque formation. These results suggest that MELT can be considered a potential therapeutic agent for targeting atherosclerotic plaque and alleviating atherosclerosis progression, providing an effective strategy for targeted atherosclerosis therapy.

The microneedles carrying cisplatin and IR820 to perform synergistic chemo-photodynamic therapy against breast cancer.

BACKGROUNDS: Surgical resection and adjunct chemotherapy or radio-therapy has been applied for the therapy of superficial malignant tumor in clinics. Whereas, there are still some problems limit its clinical use, such as severe pains and side effect. Thus, it is urgent need to develop effective, minimally invasive and low toxicity therapy stagey for superficial malignant tumor. Topical drug administration such as microneedle patches shows the advantages of reduced systemic toxicity and nimble application and, as a result, a great potential to treat superficial tumors. METHODS: In this study, microneedle (MN) patches were fabricated to deliver photosensitizer IR820 and chemotherapy agent cisplatin (CDDP) for synergistic chemo-photodynamic therapy against breast cancer. RESULTS: The MN could be completely inserted into the skin and the compounds carrying tips could be embedded within the target issue for locoregional cancer treatment. The photodynamic therapeutic effects can be precisely controlled and switched on and off on demand simply by adjusting laser. The used base material vinylpyrrolidone-vinyl acetate copolymer (PVPVA) is soluble in both ethanol and water, facilitating the load of both water-soluble and water-insoluble drugs. CONCLUSIONS: Thus, the developed MN patch offers an effective, user-friendly, controllable and low-toxicity option for patients requiring long-term and repeated cancer treatments.

The use of amphiphilic copolymer in the solid dispersion formulation of nimodipine to inhibit drug crystallization in the release media: Combining nano-drug delivery system with solid preparations.

Solid dispersion is a widely used method to improve the dissolution and oral bioavailability of water-insoluble drugs. However, due to the strong hydrophobicity, the drug crystallization in the release media after drug dissolution and the resulted decreased drug absorption retards the use of solid dispersions. It is widely known that the amphiphilic copolymer can encapsulate the hydrophobic compounds and help form stable nano-dispersions in water. Inspired by this, we tried to formulate the solid dispersion of nimodipine by using amphipathic copolymer as one of the carriers. Concerning the solid dispersions, there are many important points involved in these formulations, such as the miscibility between the drug and the carriers, the storage stability of solid dispersions, the dissolution enhancement and so on. In this study, a systemic method is proposed. In details, the supersaturation test and the glass transition temperature (Tg) measurement to predict the crystallization inhibition, the ratios of different components and the storage stability, the interactions among the components were investigated in detail by nuclear magnetic resonance (1H NMR) and isothermal titration calorimetry (ITC) and, the final dissolution and oral bioavailability enhancement. It was found that the amphiphilic copolymer used in the solid dispersion encouraged the formation the drug loading micelles in the release media and, finally, the problem of drug crystallization in the dissolution process was successfully solved.

Evaluation of anti-lipase activity and bioactive flavonoids in the Citri Reticulatae Pericarpium from different harvest time.

BACKGROUND: Inhibition of pancreatic lipase is an attractive approach to the treatment of obesity and other metabolic disorders. Naturally occurring phytochemicals are promising sources of lipase inhibitors. PURPOSE: In the present study, the anti-lipase activity of Citri Reticulatae Pericarpium (CRP) extracts was firstly evaluated in vitro. Moreover, the dynamic alteration of bioactive flavonoids in CRP collected at different time and its correlation with anti-lipase activities was investigated. STUDY DESIGN/METHODS: Quantitative analysis of multi-components by a single-marker (QAMS) method was developed and validated for simultaneous determination of six flavonoids including narirutin, hesperidin, didymin, nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone and tangeretin. Anti-lipase activity evaluation and docking studies of the flavonoids was also carried out to screen out the candidate lipase inhibitors. RESULTS: The QAMS method validation results exhibited that the developed method had desirable specificity, linearity, precision and accuracy. CRP collected in early months contained higher concentrations of bioactive flavonoids, and exhibited more potent anti-lipase activity. CONCLUSION: Harvest timing had a significant impact on the amounts of bioactive flavonoids and the anti-lipase activities of CRP extracts. The contents of total flavonoids were positively correlated with the anti-lipase activities of CRP, and polymethoxyflavones played a significant role in the hypolipidemic effect of CRP. Nobiletin might be the most potential lipase inhibitor in CRP.

[In vitro targeting effect of lactoferrin modified PEGylated liposomes for hepatoma cells].

A lactoferrin-containing PEGylated liposome system (Lf-PLS) was developed and tested in vitro as a hepatoma-targeting drug delivery system. PEGylated liposomes (PLS) were successfully prepared using the thin film hydration method with peglipid post insertion. Lf was covalently conjugated onto the carboxyl terminal of DSPE-PEG2000-COOH on liposomes. Coumarin-6 was used to trace Lf-PLS with fluorescence. The cellular uptake of this system was carried out in asialoglycoprotein receptor (ASGPR) positive HepG2 cells via confocal microscopy and flow cytometry. The Lf-PLS liposome was observed as spherical or oval vesicles with the particle size around 130 nm, zeta potential about -30 mV and encapsulation efficiency more than 80%. The confocal microscopy images and flow cytometry data demonstrated that Lf-PLS resulted in significantly higher cell association by ASGPR positive HepG2 cells compared to PLS. The association between Lf-PLS and cells were dependent on the concentration, time and temperature, which was inhibited by pre-incubation with excessive free Lf. The results suggest that Lf-PLS has a good targeting effect on HepG2 cells in vitro. The targeting mechanism may be related to the specific binding of Lf and ASGPR on HepG2 cells, which guides Lf-PLS to the cell surface to induce an active endocytosis process. All these results demonstrated that Lf-PLS might be a potential drug delivery system in targeting hepatocellular carcinoma, which deserves more research on its targeting ability, antitumor efficiency, and metabolism in vivo for treatment of hepatomacellular carcinoma.

Flurbiprofen axetil preconditioning allivates inhalation lung injury in rats / 第二军医大学学报

Objective To investigate the protective effects of flurbiprofen axetil(FA) on inhalation lung injury induced by lipopolysaccharides(LPS)inhalation in rats, so as to provide evidence for applying FA in treating inhalation lung injury in clinic. Methods A total of 96 adult male Sprague-Dawley rats were evenly randomized into four groups(n=24): saline negative control group(NS group), model group(LPS group), lipid emulsion preconditioning control group(Lip+LPS group), and FA preconditioning group(FA+LPS group). The model of inhalation lung injury was established with endotracheal instillation of LPS(1 mL/kg)in all experimental groups. NS group was identical to the other three groups except that saline(1 mL/kg)was administered instead of LPS. Lipid emulsion(20%,1 mL/kg)or FA injection(1 mL/kg,10 mg/mL)was intravenously injected via vena caudalis 1 hour before LPS in Lip+LPS and FA+LPS groups, respectively. Rats were sacrificed at 1 h, 6 h, 12 h and 24 h after LPS injection and assigned to 1 h, 6 h, 12 h and 24 h subgroups(n＝6). The arterial blood gas was analyzed and the lungs were removed for determination of the wet/dry mass(W/D)ratio and evaluation of histological injury in all groups. Real-time PCR was used to detect the mRNA levels of PPAR-α and PPAR-γ in rats lung homogenates. The concentration of tumor necrosis factor-alpha(TNF-α)in rat serum was determined by ELISA. Results The rats in LPS and Lip+LPS groups showed damaged structure of lung tissue and inflammation. The mRNA levels of PPAR-α and PPAR-γ in the lung tissues of LPS group were significantly lower than those of NS group(P< 0.05). The serum concentration of TNF-α of LPS group was significantly higher than that of NS group (P<.05). The pulmonary lesions in FA+LPS group were ameliorated compared with those in LPS and Lip+LPS groups. Pressure of oxygen in arterial blood(PaO2)was signficantly higher and semi-quantitative pathological score of lung was signficanlty lower in FA+LPS group than those in LPS and Lip+LPS group at 6 h, 12 h and 24 h after injection(P< 0.05). The mRNA levels of PPAR-α and PPAR-γ in rat lung tissues of FA+LPS group were signficanlty higher than those of LPS and Lip+LPS groups at all times, especially, at 6 h after the intravenous injection of LPS(P< 0.05). The serum concentration of TNF-α of FA+LPS group was significantly lower than that of LPS and Lip+LPS groups at all times, expecially, at 1 h after the intravenous injection of LPS (P<.05). Conclusion FA preconditioning can alleviate the inflammation and protect inhalation injury to lung tissues induced by LPS in rats, which may involve the up-regulation of PPAR-α andPPAR-γ mRNA levels in rat lung tissues and the down-regulation of serum TNF-α.

In vitro study of transdermal penetration and iontophoresis of hepatitis B vaccines through rat skin.

In vitro percutaneous delivery of hepatitis B vaccines was investigated in order to assess the penetration of vaccine under passive diffusion and iontophoresis conditions. The study was carried out using Franz vertical diffusion cell through the hairless abdominal skin of Sprague-Dawley (SD) rats. Enzyme-linked immunosorbent assay (ELISA) was used to determine the cumulative amount of permeation and the retention amount of drug in skin. Passive diffusion alone resulted in less skin permeation and retention of hepatitis B vaccines, only (2.1 +/- 0.1) ng x cm(-2) and (2.3 +/- 0.1) ng x cm(-2) after 24 h when the initial concentration of vaccine in the donor compartment was 23 microg x mL(-1) and 46 microg x mL(-1), respectively. After removing the stratum corneum, the permeation and retention amount of hepatitis B vaccines increased to (383.7 +/- 86.2) ng x cm(-2) and (16.8 +/- 4.6) ng x cm(-2), respectively, 171.6-folds and 2.1-folds more than that from its intact skin with the drug loaded at 46 microg x mL(-1). Iontophoresis induced a significant increase of cumulative and retention amount of hepatitis B vaccines through the skin (P < 0.05). Application of iontophoresis significantly enhanced the permeation of hepatitis B vaccines (P < 0.05) by 2.7-folds and 6.6-folds for the intact skin, and by 1.6-folds and 1.8-folds for the tape-stripped skin with initial drug loading of 23 microg x mL(-1) and 46 microg x mL(-1), respectively. Iontophoresis also significantly increased the amount of drug retained in the skin. After applying iontophoresis for 6 h, the amount of skin retention was nearly the same as passive diffusion for 24 h both from intact skin [(16.8 +/- 4.6) ng x cm(-2) vs (13.3 +/- 5.4) ng x cm(-2)] (P > 0.05) and tape-stripped skin [(36.7 +/- 14.1) ng x cm(-2) vs (26.8 +/- 11.2) ng x cm(-2)] (P > 0.05). Overall, these findings revealed that the transportation efficiency of bioactive substance like hepatitis B vaccines may be improved by iontophoresis, which can be potentially used in the field of transcutaneous immunization.

[Optimization of ligustrazine phosphate thermosensitive gel formulation by central composite design-response surface methodology].

OBJECTIVE: To optimize the formulation of ligustrazine phosphate thermosensitive gel by the central composite design-response surface methodology (RSM plus CCD). METHODS: In the formulation design using RSM plus CCD, independent variables were the amounts of poloxamer 407 and poloxamer 188, and gel temperature was dependent variable. Multilinear and quadratic models were used to estimate the relationship between the dependent and the independent variables, select the optimal formulations and validate. RESULTS: The quantitative relationships between two factors and evaluation index were characterized, quadratic model had better prediction capability than multilinear model. CONCLUSION: Quadratic model is performed in the optimization of formulation due to the statistical confidence. The optimization of ligustrazine phosphate thermosensitive gel formulation can be achieved by the central composite design and response surface methodology.

Increased expression of KCTD9, a novel potassium channel related gene, correlates with disease severity in patients with viral hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>Studies have shown that potassium channel plays a pivotal role in T cell activation. The expression of potassium channel gene KCTD9 was evidenced being highly upregulated in patients with severe hepatitis B (SHB). To understand this phenomenon further, tissue and cellular expression profiles of KCTD9 were investigated in patients with SHB.</p><p><b>METHODS</b>A rabbit peptide polyclonal antibody was prepared. Various samples including peripheral blood mononuclear cells (PBMCs); livers from patients with SHB or mild chronic hepatitis B, were examined for KCTD9 expression by quantitative real time PCR and immunohistochemistry staining (IHC). Confocal microscopy was used to illustrate the localizations of the expressions.</p><p><b>RESULTS</b>Increased expression of KCTD9 was observed in PBMC in over 35.7% of the patients with SHB when compared with that of patients with mild chronic hepatitis B. In all patients, the relative value of increased KCTD9 mRNA was positively correlated with alanine aminotransferase, aspartate aminotransferase, total bilirubin and direct bilirubin but negatively with serum albumin. The expression was mainly located in hepatocytes, bile duct epithelial cells, Kupffer cells and inflammatory cells, and in the cytoplasm of PBMCs from the healthy individuals and patients with mild chronic hepatitis B, whereas in both cytoplasm and nuclei in those from patients with SHB.</p><p><b>CONCLUSION</b>The increased expression of potassium channel gene KCTD9 correlates with disease severity in patients with viral hepatitis B.</p>

Effect of puerarin and ligustrazine on cultured hippocampal neurons injury induced by Abeta25-35 / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the changes of MDA, SOD, LDH of cultured hippocampal neurons injury induced by amyloid-beta protein (Abeta 25-35) and the protective effect of puerarin and ligustrazine.</p><p><b>METHOD</b>Primary hippocampal neurons were cultured and induced by Abeta 25-35. The concentrations of MDA, SOD and LDH in cultured hippocampal neurons were measured after exposed to Abeta 25-35, puerarin and ligustrazine.</p><p><b>RESULT</b>The Alzheimer disease (AD) model was successfully established in cultured hippocampal neurons. AD group has remarkably increased MDA and LDH level, and decreased SOD level, Piracetan group and combined application group of have remarkably decreased MDA and LDH level and increased SOD level, compared with AD group (P < 0.01). Ligustrazine together with puerarin group has remarkably decreased MDA and LDH level and increased SOD level, compared with ligustrazine group and puerarin group (P < 0.05).</p><p><b>CONCLUSION</b>Abeta 25-35 can induce cultured hippocampal neurons injury, combined application of ligustrazine, and puerarin can alleviate the injury.</p>