Catechin-loaded Eudragit microparticles for the management of diabetes: formulation,characterization and in vivo evaluation of antidiabetic efficacy

Catechin (CT) is natural molecule proved for antidiabetic activity. Clinical application of CT is highly restricted because of its low bioavailability and ineffectiveness in in vivo conditions. Therefore, the main objective of the present investigation was to formulate CT-loaded Eudragit RS 100 microparticles and evaluated for its potential against diabetes. CT microparticles showing highest entrapment efficiency of 92.3?±?6.5% and higher percentage yield of 63.46?±?4.3% was selected as optimised formulation. CT microparticles treated rats showed significantly lower blood glucose, cholesterol, LDL, free fatty acid and triglyceride concentrations in comparison to pristine CT-treated rats. The glucose and lipid profiles of microparticle formulation were akin to normal rats. Moreover, CT microparticles did not produce obesity even after 60 days which is a comment side effect of antidiabetic drugs. These results indicate that the CT microparticles can be applied as potential and safe carrier for the treatment of diabetes.     

A Comparison of Aerosolization and Homogenization Techniques for Production of Alginate Microparticles for Delivery of Corticosteroids to the Colon

Alginate microparticles incorporating hydrocortisone hemisuccinate were produced by aerosolization and homogenization methods to investigate their potential for colonic drug delivery. Microparticle stabilization was achieved by CaCl₂ crosslinking solution (0.5 M and 1 M), and drug loading was accomplished by diffusion into blank microparticles or by direct encapsulation. Homogenization method produced smaller microparticles (45-50 μm), compared to aerosolization (65-90 μm). High drug loadings (40% wt/wt) were obtained for diffusion-loaded aerosolized microparticles. Aerosolized microparticles suppressed drug release in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) prior to drug release in simulated colonic fluid (SCF) to a higher extent than homogenized microparticles. Microparticles prepared using aerosolization or homogenization (1 M CaCl₂, diffusion loaded) released 5% and 17% of drug content after 2 h in SGF and 4 h in SIF, respectively, and 75% after 12 h in SCF. Thus, aerosolization and homogenization techniques show potential for producing alginate microparticles for colonic drug delivery in the treatment of inflammatory bowel disease.     

Effects of anti‐TNF‐α agents on circulating endothelial‐derived and platelet‐derived microparticles in psoriasis

Psoriasis involves TNF‐α secretion leading to release of microparticles into the bloodstream. We investigated the effect of TNF blockers on microparticles levels before and after treatment in patients (twenty treated by anti‐TNF‐α agents and 6 by methotrexate) with severe psoriasis. Plasmatic microparticles were labelled using fluorescent monoclonal antibodies and were analysed using cytometry. Three months later, 70% of patients treated with anti‐TNF‐α agents achieved a reduction in PASI score of at least 75%. The clinical improvement in patients treated with anti‐TNF‐α agents was associated with a significant reduction of the mean number of platelet microparticles (2837/μl vs 1849/μl, P = 0.02) and of endothelial microparticles (64/μl vs 22/μl, P = 0.001). Microparticles are significantly decreased in psoriatic patients successfully treated by anti‐TNF‐α. Microparticles levels as circulating endothelial cells represent signs of endothelial dysfunction and are elevated in psoriasis. Then, TNF blockade may be effective to reduce cardiovascular risk through the reduction of circulating microparticles.     

幽门螺杆菌根除前后对CagA~+冠心病患者血管内皮功能的影响

目的:探讨幽门螺杆菌(Hp)根除前后细胞毒素相关基因A阳性(CagA+)对冠状动脉粥样硬化性心脏病(冠心病)患者血管内皮功能的影响。方法:选择Hp+CagA+冠心病患者57例为根除治疗组,同期住院的57例Hp-CagA-冠心病患者为对照组。在常规治疗的基础上,根除治疗组接受根除Hp治疗,对照组接受安慰剂治疗,研究期为6个月。两组研究对象在实验前后接受血脂、外周血循环内皮微颗粒(cEMP)水平及肱动脉对反应性充血的内皮依赖血管扩张反应(FMD)检测。结果:治疗后根除治疗组患者的血浆总胆固醇、cEMP水平比治疗前明显降低,FMD明显升高(P均<0.05)。治疗前根除治疗组总胆固醇高于对照组(P<0.01),治疗后两组总胆固醇比较差异无统计学意义(P>0.05)。结论:根除Hp+CagA+,可改善合并Hp+CagA+感染冠心病患者的血管内皮功能。     

Spray drying from organic solvents to prepare nanoporous/nanoparticulate microparticles of protein: excipient composites designed for oral inhalation

Objectives The aim of this study was to determine if spray‐drying could successfully produce microparticles containing the model protein trypsin in a form suitable for inhalation. Methods Trypsin was spray‐dried with raffinose from a methanol : n‐butyl acetate solvent system (MeOH : BA). The solvent system was then adjusted to include water, and trypsin was co‐spray‐dried with raffinose, trehalose or hydroxpropyl‐β‐cyclodextrin. The spray‐dried products were characterised by SEM, XRD, DSC, TGA and FTIR. Protein biological activity and in‐vitro deposition of trypsin : excipient nanoporous/nanoparticulate microparticles (NPMPs) was also assessed. Key findings The inclusion of water in a MeOH : BA solvent system allowed for the successful production of NPMPs of trypsin : excipient by spray‐drying. Trypsin formulated as trypsin : excipient NPMPs retained biological activity on processing and showed no deterioration in activity or morphological characteristics when stored with desiccant at either 4 or 25°C. Hydroxpropyl‐β‐cyclodextrin showed advantages over the sugars in terms of producing powders with appropriate density and with greater physical stability under high‐humidity conditions. Fine particle fractions of between 41 and 45% were determined for trypsin : excipient NPMPs. Conclusions NPMPs of trypsin : excipient systems can be produced by spray‐drying by adjustment of the solvent system to allow for adequate solubility of trypsin.     

Microparticles in newborn cord blood: slight elevation after normal delivery

Introduction

Microparticles formed during delivery may add to the well functioning hemostasis, but also to hypercoaguability in the newborn.We wanted to investigate whether microparticles in newborn cord plasma differ from those in adult plasma in terms of concentration, procoagulant activity, and effect on thrombin generation.

Materials and Methods

Three different techniques were used to analyze microparticles. To enumerate and characterize microparticles, flow cytometry and ELISA, based on the prothrombinase reaction, were used. The effect of microparticles derived tissue factor on thrombin generation was measured indirectly by Calibrated Automated Thrombography in newborn cord and adult platelet free plasma.

Results

The flow cytometric measurements of microparticles showed no significantly increased microparticle concentration in newborn cord compared with adult plasma. By the use of ELISA a significantly increased procoagulant activity of microparticles was found in newborn cord plasma as compared to adult plasma. Initiation of thrombin generation by adding phospholipids alone suggested a higher microparticle activity in newborn cord plasma than in adult plasma.

Conclusions

Our results show a higher impact of microparticles on the hemostatic system in newborn cord plasma than in adult plasma in terms of activity, but not concentration. Calibrated Automated Thrombography and ELISA suggest an increased microparticle activity in newborn cord plasma, but comparable results in microparticle number as determined by flow cytometry argue against strong platelet activation during birth.     

Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay

Introduction

Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification.

Materials and Methods

Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated.

Results

Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV + MPs (P = 0.002) and platelet-derived MPs (PMPs) (P = 0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV + MPs (P = 0.0004) and PMPs (P = 0.0004). A single freeze thaw cycle of samples led to an increase in AnnV + MPs (P = 0.0020) and PMPs (P = 0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels.

Conclusions

This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease.     

胃癌患者内皮细胞微颗粒的检测及其意义

目的比较胃癌患者与正常对照组血浆中内皮细胞微颗粒（EMPs）、血管性血友病因子（vWF）水平的变化,探讨EMPs检测在胃癌中的意义。方法采用流式细胞术及酶联免疫吸附试验（ELISA）检测68例胃癌患者和20例正常对照者血浆中EMPs、vWF的水平,分析这些指标与临床特征的关系。结果胃癌患者血浆vWF及EMPs水平显著高于正常对照组（P〈0.01）,血浆vWF及EMPs水平Ⅲ、Ⅳ期患者高于Ⅰ、Ⅱ患者（P〈0.01）,有淋巴结及肝转移者高于无淋巴结及肝转移者（P〈0.01）,有静脉浸润者高于无静脉浸润者（P〈0.01）,浸润至肌层、浆膜层者高于浸润至黏膜或黏膜下层者（P〈0.01）。结论血浆EMPs可准确反映内皮功能,内皮功能紊乱参与胃癌的转移过程,EMPs可作为判断胃癌的病情、预后、疗效的指标之一。     

内皮细胞微粒对人脐静脉内皮细胞VCAM-1和ICAM-1表达的影响

目的:探讨内皮细胞微粒(Endothelial microparticles,EMPs)对人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)血管细胞粘附分子-1(Vascular cellular adhesion molecule-1,VCAM-1)和细胞间粘附分子-1 (Intercellular adhesion molecule 1,ICAM-1)表达的影响.方法:取生长良好的第4、5代HUVECs,将细胞以1×105个/ml密度接种于5 cm2的培养皿中,待细胞生长近80％融合时进行干预.按0、1×102、1×103、1×104、1×105个ml浓度的EMPs进行分组及刺激,每组12个样本.在共同培养24h后收集细胞.分别采用实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测VCAM-1和ICAM-1 mRNA和蛋白的表达.结果:HUVECs受EMPs刺激,VCAM-1和ICAM-1 mRNA及蛋白表达显著增加,且EMPs的这种作用呈浓度依赖性增强(均P＜0.05).结论:EMPs能上调HUVECs VCAM-1和ICAM-1的表达,EMPs不仅是内皮功能障碍的标记物,还能加重内皮功能损害.