雌二醇聚乳酸类微囊的制备与体外评价

目的　以聚乳酸或乳酸 乙醇酸共聚物为载体材料制备雌二醇缓释微囊。方法　实验中采用乳化 溶剂萃取法制备微囊 ,考察了影响微囊特性以及体外释药的各种因素 ,采用单因素试验方差分析进行因素影响的显著性检验 ,并在此基础上采用T方法进行两两间多重比较 ,进一步考察因素各水平间差异的显著性。结果　结果表明 ,粒径和加水萃取速度对雌二醇微囊的包封率有显著性影响 (P <0 0 1) ;随油相载体的浓度以及加水萃取速度对微囊的粒径影响显著 (P <0 0 1)。体外释药研究表明 ,加水萃取速度和载体的型号和分子量对微囊释药影响显著 (P <0 0 1)。但这些影响因素的各水平之间的差异性并不都具有显著性。结论　通过改变处方以及制备工艺可以得到具有不同性质的雌二醇聚乳酸类缓释微囊     

PLGA黏度及PVA浓度对微球形成过程中物理性质的影响

采用复乳.溶剂蒸发法制备乳酸.羟基乙酸共聚物(PLGA)微球,并用激光粒度仪测定制备过程中乳滴的体积平均粒径、粒径分布(d_(0.1)、d_(0.5)、d_(0.9))和重量比表面积(SSA)等物理性质的动态变化,考察PLGA特性黏度及聚乙烯醇(PVA)浓度对上述性质的影响.结果表明,在0.5%PVA的体系中,乳滴的平均粒径在60 S内均呈线性急剧下降,SSA迅速增加,粒径下降速度随PLGA黏度的增加而减慢,但5 min后高黏度PLGA体系的粒径升高,SSA减小.增加外水相PVA浓度至2.0%,则高黏度PLGA体系乳滴的物理性质变化趋势与低、中黏度PLGA体系相似.     

电纺参数对PLGA/HA复合支架纤维形貌和直径的影响

目的通过静电纺丝的方法制备PLGA/HA复合支架,探讨电纺参数对复合支架纤维形貌和直径的影响。方法以三氯甲烷和N,N-二甲基甲酰胺为混合溶剂制备PLGA/HA纺丝液,通过调节PLGA的浓度、电压、接收距离,制备具有不同表面形貌的PLGA/HA复合纤维,采用SEM观察PLGA的浓度、电压、接收距离对纤维形貌和直径的影响。结果复合纤维的直径随PLGA浓度的增加而增加;随电压的增加而增加;随接收距离的增加先减小后增加。结论制备PLGA/HA复合支架较合适的电纺参数为：PLGA浓度25%,电压20KV,接收距离15cm。静电纺丝法制得的PLGA/HA复合支架有可能作为骨组织再生的支架在组织工程领域发挥作用。     

复乳法制备胰岛素PLGA纳米粒影响包封率因素考察

以Poloxamer188为乳化剂，乙酸乙酯为有机溶剂，采用复乳法制备了胰岛素乳酸／羟基乙酸共聚物（PLGA）纳米粒，考察了乳化剂和PLGA的浓度。内水相中胰岛素的浓度为pH，溶剂挥发方法和内水相中加入聚乙烯醇（PVA）等实验各因素对胰岛素PLGA纳米粒包封率的影响。结果表明，乳化剂的浓度较高，PLGA的浓度较小，内水相的pH接近胰岛素的pI(5．3），胰岛素的浓度较低，缩短有机溶剂挥发时间及内水相中加入PVA有利于提高胰岛素的包封率，经实验条件优化后制备的胰岛素PLGA纳米纳平均粒径为149．6nm，多分散性系数小于0．1，包封率提高到42．8％。     

Biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres for sustained release of risperidone: Zero-order release formulation

The preparation and investigation of sustained-release risperidone-encapsulated microspheres using erodible poly(D, L-lactide-co-glycolide) (PLGA) of lower molecular weight were performed and compared to that of commercial Risperdal Consta^? for the treatment of schizophrenia. The research included screening and optimizing of suitable commercial polymers of lower molecular weight PLGA50/50 or the blends of these PLGA polymers to prepare microspheres with zero-order release kinetics properties. Solvent evaporation method was applied here while studies of the risperidone loaded microsphere were carried out on its drug encapsulation capacity, morphology, particle size, as well as in vitro release profiles. Results showed that microspheres prepared using 50504A PLGA or blends of 5050-type PLGAs exerted spherical and smooth morphology, with a higher encapsulation efficiency and nearly zero-order release kinetics. These optimized microspheres showed great potential for a better depot preparation than the marketed Risperdal Consta^?, which could further improve the patient compliance.     

The mechanism of surface-indented protein-loaded PLGA microparticle formation: the effects of salt (NaCl) on the solidification process

The purpose of this study was to evaluate ovalbumin (OVA) leakage pathways and to explore the mechanism of the surface-indented microparticle formation in the preparation of OVA-loaded microparticles. OVA-loaded poly (D,L-lactic-co-glycolic acid) (PLGA) microparticles were prepared by a water-in oil-in water (w/o/w) solvent evaporation method associated with varied NaCl (NaCl) concentrations and adjusted with urea at 1240?mOsm?kg^?1 in the external aqueous phase. To evaluate dichloromethane (DCM)-related OVA leakage, three stirring rates, 600, 800, 1000?rpm at 25° C were carried out during the solvent evaporation stage. Both DCM and OVA levels in the external phase medium and total dispersion were sampled and measured. The time course of particle characteristics was evaluated by microscopy or SEM photography. The surface adsorptive capacities of the prepared microparticles were measured by using bovine serum albumin conjugated with fluorescein isothiocyanate (FITC-BSA). The findings were that the DCM-related OVA leakage accounted for ～34% of the total leakage. By combining NaCl in the external phase, a faster solidifying crust-like structure was formed as a barrier to remarkably reduce OVA loss and improve OVA content from 40.1 to 72.8?µg?mg^?1. The yield and OVA content for formulations containing NaCl were much improved by the ionic effect, in addition to the osmotic effect. The total entrapment efficiency was also highly increased from 43 to 72%. The formations of the crust-like surface structure of the microparticle were affected by entrapped drugs, salt content in the external phase and aqueous volume in the inner phase. A scheme was proposed to interpret the formation mechanism of the surface-indented microparticles. In comparison to the surface-smooth microparticles, the surface adsorptive capacities of the surface-indented microparticles were highly improved from 26.6 to 87.0%, determined by the adsorption of FITC-BSA.     

The Influence of Surfactant on PLGA Microsphere Glass Transition and Water Sorption: Remodeling the Surface Morphology to Attenuate the Burst Release

Purpose The stability of protein unloaded and loaded poly(lactic-co-glycolic acid) (PLGA) microspheres fabricated with surfactant was challenged through exposure to environmental conditions of different relative humidity. Methods Polyvinyl alcohol (PVA) or Triton X-100 was added to the primary emulsion of the double-emulsion solvent evaporation technique. After storage at ambient humidity and 75% relative humidity, the mechanical stability of the polymer was tested to reveal PLGA chain mobility using differential scanning calorimetry. Subsequent surface modifications were examined by atomic force microscopy (AFM), and protein release profiles were collected. Results Residual amounts of PVA and particularly Triton X-100 raised the hydrophilicity of the microspheres. When exposed to ambient humidity or 75% relative humidity, PVA and Triton X-100 had, respectively, an antiplasticizing and a plasticizing effect upon PLGA, and both led to physical aging. The high-resolution AFM imaging of microspheres containing model protein and Triton X-100 showed that the depth of the surface pores was reduced when exposed to 75% relative humidity, and the initial burst release subsequently decreased. Conclusion These studies suggested that the mechanical stability of PLGA was influenced by the addition of surfactants, which, depending on the formulation, led to surface pore remodeling under high humidity, reducing the initial burst release while maintaining the spherical integrity of the microsphere.     

不同表面活性剂修饰的汉防己甲素PLGA纳米粒制备表征及体外评价

目的:基于纳米粒的递送系统,以改善天然化合物汉防己甲素对肺癌的功效。方法:选取聚乙烯醇、普朗尼克-F127和双十二烷基二甲基溴化铵3种不同的稳定剂,采用单乳化扩散溶剂挥发法制备载汉防己甲素的PLGA纳米粒,考察不同稳定剂对载药纳米粒粒径、ζ电位以及对肺癌A549细胞摄取的影响。结果:2%,1%和0.1%浓度的PVA、PF127和DMAB制备的纳米粒呈表面光滑、大小均一的球形,粒径范围均控制在180~200nm;药物包封率为50%~60%;体外释放实验显示,在pH 7.4的PBS溶液中3组载药纳米粒均呈现缓慢持续释药;细胞学实验结果表明3组纳米粒给药系统均表现出比药物更强的抗肿瘤活性。结论:对PLGA进行表面修饰制备的纳米载体能使汉防己甲素的给药效率得到明显提升。     

Effects of Sugars and Polymers on Crystallization of Poly(ethylene glycol) in Frozen Solutions: Phase Separation Between Incompatible Polymers

Purpose. This study examined the effect of third components (low-molecular-weight saccharides and polymers) on the crystallization of poly(ethylene) glycol (PEG) in frozen solutions, focusing on the relationship between their crystallization-inhibiting ability and molecular compatibility. Methods. Effects of sugars and polymers on the crystallization of PEG 3000 in frozen solution were monitored by differential scanning calorimetry (DSC). Pulsed-NMR was employed to monitor the molecular mobility of water and solutes in the frozen solutions. Miscibility between PEG and third components in aqueous solution was estimated from the lowering of cloud point of PEG 20,000. Thermal analysis of frozen solutions containing some non-crystallizing solutes was used to examine the possibility of phase separation in frozen solutions. Results. Some sugars and polymers inhibited the crystallization of PEG and formed practically stable amorphous phases among ice crystals. The mobility of solute molecules in the amorphous phase increased above the softening temperature of maximally concentrated solutions (T_s), whereas that of water molecules appeared at a lower temperature. Mono- and disaccharides that are relatively less miscible with PEG in solution inhibit PEG crystallization to a lesser degree. Two T_s regions were observed in frozen solutions containing both polyvinylpyrrolidone (PVP) and dextran, at much lower concentrations than those causing aqueous two-phase separation at ambient temperatures. Conclusions. Ice crystallization raises the concentration of solutes in the remaining solution, which can lead to phase separation in the amorphous phase. Molecular compatibility between components is an important factor determining their propensity to phase separate and crystallize.     

Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes IV: An Index of Chromosomal Damage in the Rhesus Monkey (Macaca mulatta)

We report evaluation in rhesus monkeys of a flow cytometricprocedure (MicroFlow) that has previously been shown to allowassessment of micronucleated reticulocytes (MN-RETs) in theperipheral blood of rats and dogs. Reticulocytes (RETs) werelabeled with anti-CD71-fluorescein isothiocyanate, DNA was stainedwith propidium iodide using RNase treatment, and anti-CD61-phycoerythrinwas used to reduce interference from platelets. Flow cytometricdata were compared with microscopic scores of peripheral bloodand bone marrow using standard acridine orange staining. A singleiv administration of cyclophosphamide (CP, 5 mg/kg) inducedan approximately 10-fold increase in blood MN-RET frequency,with the peak occurring 2 days after administration. After dailyCP treatment to approximate a steady-state condition, the frequencyof MN-RETs in peripheral blood was approximately 25% of thatin bone marrow, indicating strong selection against MN-RETs.Nonetheless, CP-treated animals exhibited markedly elevatedblood MN-RET values (2.45–3.99%, n = 3; compared to amean baseline of 0.12%, n = 6). These measurements closely reflectedthe increased frequencies observed in the bone marrow compartment(Spearman correlation coefficient = 0.9856, n = 6). These datasuggest that MN-RET measurements in blood are suitable for assessingchemical-induced chromosomal damage and can be readily integratedinto routine toxicity tests, allowing genotoxicity data to beobtained as an integral part of toxicity evaluations. Microscopy-basedscoring is challenging due to the low frequency of RETs andMN-RET in monkeys, but sufficient numbers of cells are easilyscored with the flow cytometric procedure.     

Influence of Particle Size,Air Flow,and Inhaler Device on the Dispersion of Mannitol Powders as Aerosols

Purpose. To study the effect of particle size, air flow and inhaler type on the dispersion of spray dried mannitol powders into aerosols. Methods. Mannitol powders were prepared by spray drying. The solid state properties of the powders were determined by laser diffraction, X-ray powder diffraction, scanning electron microscopy, freeze fracture, Karl Fischer titration and gas pycnometry. The powders were dispersed using Rotahaler^® and Dinkihaler^®, connected to a multistage liquid impinger at different air flows. Results. Three crystalline mannitol powders with primary particle size (MMD) 2.7, 5.0, 7.3 m and a similar polydispersity were obtained. The particles were spherical with a density of 1.5 g/cm³ and a moisture content of 0.4 wt.%. At an air flow of 30 L/min all the powders were poorly dispersed by both inhalers. With the Rotahaler^® increasing the flow (60–120 L/min) increased the fine particle fraction (FPF) in the aerosols for the 2.7 m powder, and decreased the FPF for the 7.3 m powder; whereas the FPF for 5.0 m powder was unaffected. With the Dinkihaler^®, all the powders were near complete dispersion at 60 L/min. Conclusions. The FPF in the mannitol powder aerosols was determined by an interplay of the particle size, air flow and inhaler design.     

Effects of polymerization variables on PLGA properties: molecular weight, composition and chain structure

In order to study the influence of the variables which control the properties of polylactic-glycolic acid two experiments were conducted according to appropriate experimental designs. The responses studied were yield, weight average molecular weight (GPC), relative proportion of lactic and glycolic acids in the polymer (¹H-NMR) and the ratio of lactic-glycolic to glycolic-glycolic units (¹³C-NMR). The variables included in the first study were the relative proportion of lactide and glycolide in the mixture and the concentrations of lauryl alcohol and stannous octoate; the results showed a statistically significant influence of the relative proportions of lactide and glycolide in the mixture and the concentration of lauryl alcohol on the lactic/glycolic proportion in the polymer. The second study involved time, temperature and lauryl alcohol concentration as variables. The findings showed a statistically significant negative effect of temperature on weight average molecular weight and of temperature, time and time-temperature interaction on yield. Ratios between lactic-glycolic and glycolic-glycolic units ranged from 1:1 to 1:4. None of the variables studied had any effect on this characteristic.     

Poly(lactic-co-glycolic acid) nanoparticles for sustained release of allyl isothiocyanate: characterization,in vitro release and biological activity

The objective of this study is to establish the ability of entrap allyl isothiocyanate (AITC) into polymeric nanoparticles to extend its shelf life and enhance its antiproliferative properties. Natural compounds, such as AITC, have showed multi-targeting activity resulting in a wide-range spectrum of therapeutic properties in chronic and degenerative diseases, conversely with most current pharmaceutical drugs showing single targeting activity and often result in drug resistance after extended administration periods. Apparently, AITC-loaded poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) reduced AITC degradation and volatility and were able to extend AITC shelf life compared with free AITC (65% vs. 20% in 24?h, respectively). Cell viability and uptake of AITC-loaded nanoparticles were studied in vitro, showing that the protection and sustained release of AITC from polymeric NPs involved a larger toxicity of tumoral cells. These nanoparticles could be used as protective systems for enhancing a biological activity.     

双炔失碳酯、屈洛昔芬、诺美孕酮及米非司酮对离体培养大鼠黄体细胞凋亡的影响(英文)

目的:研究双炔失碳酯、屈洛昔芬、诺美孕酮和米非司酮是否可诱导离体培养的大鼠黄体细胞凋亡,方法:离体培养的大鼠黄体细胞经各种药物作用后HE染色,观察其形态学变化,抽提细胞DNA进行琼脂糖凝胶电泳,流式细胞仪定量分析凋亡细胞数,结果:四种药物均降低细胞存活率,屈洛昔芬诱导细胞凋亡,其它三种药物引起细胞坏死.细胞经屈洛昔芬作用后发生DNA断裂,电泳后显示典型的“DNA ladder",屈洛昔芬1.25,2.5或3.75 mg·L~(-1)分别引起15.4％,75.4％或90.5％的细胞凋亡,结论:屈洛昔芬诱导离体培养的大鼠黄体细胞凋亡,而双炔失碳酯、诺美孕酮和米非司酮诱导细胞坏死。     

Cystatin incorporated in poly(lactide-co-glycolide) nanoparticles: development and fundamental studies on preservation of its activity

Preservation of biological activity is still a major challenge for successful formulation and delivery of protein drugs. Cystatin, a potential protein drug in cancer therapy, was incorporated in poly(lactide-co-glycolide) nanoparticles by the water-in-oil-in-water emulsion solvent diffusion technique. In order to preserve the biological activity of cystatin, a specific modification of the method of producing nanoparticles was introduced. The activity of cystatin was strongly influenced by the stirring rate during preparation and, to a lesser extent, by selected organic solvents. A synergistic effect of mechanical stirring and sonication, both at low energy levels, enabled nanoparticles to be formed without denaturing the cystatin. Nanoparticles produced by the optimised method ranged from 300 to 350 nm in diameter with 85% of the starting cystatin activity. The loading efficiency of cystatin depends on polymer type and ranged from 12 to 57%, representing an actual loading of 0.6-2.6% (w/w). Among various cryo-/lyoprotectants bovine serum albumin was identified as the most successful. The use of a protein protectant prior to nanoparticle formation was essential to maintaining the biologically active three-dimensional structure of cystatin. In addition, a specific type of poly(lactide-co-glycolide) polymer, particularly in terms of its functional groups, was identified to be important in retaining cystatin activity. Cystatin incorporated into nanoparticles in this way maintains its structural integrity, making it suitable for effective drug delivery.     

流式细胞术和平板计数法用于地衣芽孢杆菌活菌制剂检测的比较研究

目的 考察流式细胞技术和平板计数法2种方法检测地衣芽孢杆菌活菌制剂（片剂、颗粒剂和胶囊剂）中芽孢数的不同。方法 分别采用BD FACSMicroCount全自动微生物分析系统和平板计数法检测不同水浴温度及不同水浴时间处理过的地衣芽孢杆菌活菌胶囊中的芽孢数。同时采用上述2种方法检测3种地衣芽孢杆菌活菌制剂（片剂、颗粒剂和胶囊剂）中的活菌数、芽孢数和芽孢率。结果 流式细胞技术和平板计数法对于检测不同水浴温度及不同水浴时间处理过的地衣芽孢杆菌活菌胶囊中的芽孢数无显著性差异。采用上述2种方法检测的3种地衣芽孢杆菌活菌制剂（片剂、颗粒剂和胶囊剂）中的活菌数、芽孢数和芽孢率的结果比较均无显著性差异。结论 流式细胞技术和平板计数法对于3种地衣芽孢杆菌活菌制剂（片剂、颗粒剂和胶囊剂）检测活菌数、芽孢数和芽孢率无显著性差异。     

Effects of Indometacin,Naproxen and Paracetamol on Regional Blood Flow in Rabbits: A Microsphere Study

Abstract: Ulcerogenic side-effects and prostaglandin-synthesis-inhibiting capacity are well documented in indomethacin treatment. According to recent works, indomethacin reduces gastrointestinal blood-flow. Naproxen and paracetamol, claimed to be prostaglandin-synthesis-inhibitors, have few ulcerogenic side effects. In an attempt further to study the indomethacin effects and to reveal whether naproxen and paracetamol have similar effects, the labelled microsphere technique was used. The regional blood flow determinations were made before, and 12–15 min. after, the injection of the drugs. Indomethacin 3 mg/kg, reduced gastrointestinal blood flow and increased arterial blood flow to the liver. Naproxen, 10 mg/kg, and paracetamol, 25 mg/kg, had no effects except for a very small decrease in liver blood flow with paracetamol. The results strongly suggest that, at least under light general anaesthesia, prostaglandins influence resting blood flow in the gastrointestinal tract, the liver and parts of the brain. The results more raise doubts whether naproxen and paracetamol inhibit prostaglandin synthesis in these tissues. These data offer a plausible explanation as to why naproxen and paracetamol are usually well tolerated in the gastrointestinal tract. None of the drugs tested influenced resting blood flow in muscles, tendons, bones, joints or synovial membranes.     

Effect of Bioflavonoids (Trihydroxyethylrutin and Disodium Flavodate) in vitro on Neutrophil Reactive Oxygen Production and Phagocytic Ability Assessed by Flow Cytometry

SUMMARY

Neutrophil granulocytes have been described as agents of defence and destruction. The effect of two flavonoid compounds (trihydroxyethylrutin and disodium flavodate) on the phagocytic ability and generation of reactive oxygen radicals of neutrophils was studied at concentrations of 5?mg/l, 50?mg/l and 100?mg/l. Flow cytometry was used to study phagocytic ability by measuring uptake of fluorescein-labelled bacteria. The generation of reactive oxygen intermediates was estimated by means of a CD16 phycoerythrin-conjugated mouse anti-human monoclonal antibody. In vitro trihydroxyethylrutin (THET) and disodium flavodate (DF) treatment reduced reactive oxygen production (DF at 5?mg/l–40%, at 50?mg/l–71% and at 100?mg/l–82%; THET at 5?mg/l–53%, at 50?mg/l–88%, at 100?mg/l–93%; all p?<?0.001). This was rapidly reversible after plasma exchange.

Both flavonoids did not affect neutrophil phagocytic ability.

We conclude that THET and DF could decrease oxidative tissue damage by neutrophils. A beneficial effect in peripheral vein disease could be anticipated from these results.     

无蹼壁虎抗肿瘤活性成分对HepG2细胞增殖、迁移及凋亡的影响

目的观察无蹼壁虎抗肿瘤活性成分(Gekko Swinhonisanti-neoplasm active component,GSAAC)对人肝癌HepG2细胞增殖、迁移及凋亡的影响。方法 GSAAC与体外培养的人肝癌HepG2细胞共培养,分别以MTT法和Transwell小室检测其对细胞增殖及迁移、侵袭的影响;免疫组化法检测PCNA的表达;Hochest33342荧光染色法、TUNEL法观察GSAAC对HepG2细胞的作用;流式细胞术检测细胞周期及早期凋亡率。结果 GSAAC(25～400 mg.L-1)可明显抑制HepG2细胞的增殖、迁移和侵袭能力,呈浓度依赖性;Ho-chest33342、TUNEL染色及流式细胞术结果显示,GSAAC可诱导细胞发生早期凋亡,阻滞HepG2细胞从S期进入G2期。结论 GSAAC可能通过影响细胞周期进而抑制HepG2细胞增殖和迁移并诱导细胞发生凋亡,进而实现抑制肿瘤的作用。     

DNA/polyethylenimine transfection particles: Influence of ligands, polymer size, and PEGylation on internalization and gene expression

Receptor-binding ligands have been incorporated into DNA/polyethylenimine (PEI) complexes to enhance cell binding and cellular internalization. This study characterizes receptor-mediated uptake of DNA/PEI complexes on a cellular basis. A novel assay based on flow cytometry was applied, discriminating between total cell-associated and extracellularly bound DNA complexes. Receptor-mediated uptake of ligand-containing DNA/PEI (molecular weight, 800 kd) complexes was found to occur quickly (within 1 hour), whereas unspecific uptake through adsorptive endocytosis is less efficient or requires extended periods to reach the same degree of internalization. Rapid, receptor-mediated internalization requires a small complex size; however, large, aggregated complexes show higher gene expression. Using PEI 25 kd conjugated to large proteins such as transferrin or antibodies, improper condensation with DNA leads to suboptimal uptake and gene expression, whereas partial replacement of ligand-PEI with unconjugated PEI increases both uptake and transfection. In contrast, the 8 kd protein epidermal growth factor conjugated to PEI 25 kd properly condenses DNA and mediates specific uptake into human adenocarcinoma (KB) cells. Modification of the complex surface with appropriate amounts of poly(ethylene glycol) (PEG) does not block ligand-mediated internalization. A higher degree of PEGylation reduces the internalization of transferrin or antibody-containing complexes to a level similar to that of ligand-free complexes. In contrast, epidermal growth factor-mediated uptake is less effected by excessive PEGylation.