Water-in-Oil Microemulsions Containing Medium-chain Fatty Acids/Salts: Formulation and Intestinal Absorption Enhancement Evaluation

Purpose. Water-in-oil (w/o) microemulsions have been developed which, in addition to non-ionic medium-chain glycerides, incorporate ionic lipids, primarily medium-chain fatty acids, such as caprylic (C₈) capric (C₁₀) and lauric (C₁₂) acids and their corresponding sodium salts. The absorption enhancing activity of w/o microemulsions incorporating these lipids was evaluated in the rat using Calcein (MW = 623) a water-soluble and poorly absorbed marker molecule. Methods. Phase diagrams were constructed where C₈/C₁₀ or C₁₂ fatty acids were treated as lipophilic surfactants and their sodium salts as hydrophilic ones. The anesthetised rat model was employed to evaluate Calcein absorption upon a single intraduodenal administration from a solution and the various w/o microemulsions. Results. A wide range of clear and transparent w/o microemulsions were obtained at ambient temperature either in liquid or solid form when a fixed blend of medium chain fatty acid/salt was titrated by a fixed ratio of the oil containing the oil-soluble mono- and diglycerides and deionized water or physiological saline. Upon intraduodenal administration in the anesthetised rat, the absorption of Calcein was improved from about 2% in aqueous solution up to about 37% in w/o microemulsions. Solid and liquid formulations were equally effective in improving bioavailability. The absorption enhancement activity of the fatty acids/salts followed the order C₈ C₁₀ > C₁₂. Absorption enhancement of Calcein was significantly reduced in the absence or presence of low levels of C₈/C₁₀ mono-/diglycerides. Conclusions. These results further support the use of medium-chain glycerides and fatty acids/salts in microemulsion formulations to improve intestinal absorption of water-soluble compounds.     

Quantitative Solubility Relationships and the Effect of Water Uptake in Triglyceride/Monoglyceride Microemulsions

Purpose This paper aims to elucidate quantitative relationships between small molecule solubility/water-uptake in triglyceride/monoglyceride lipid formulations, the chemical structure of the solute, and the solvent composition. Methods Solubility and water uptake in tricaprylin/1-monocaprylin and tricaprylin/1-monocaprin mixtures in the “microemulsion” region at 37°C were determined with HPLC and KF coulometry, respectively. Twelve model solutes varying in hydrogen bond acidity, basicity, polarity, and molecular volume were chosen. Linear free energy relationships (LFER) (Abraham type) were implemented to obtain solvent coefficients at various monoglyceride concentrations. Results Profiles for both solubility and water uptake (at different water activities) in lipid mixtures containing different monoglycerides were superimposable, producing a single master curve when the monoglyceride concentrations were plotted on a molar scale. The LFER derived solvent coefficients showed a systematic dependence on the lipid composition consistent with the view that relative solubility is determined largely by the molar concentrations of individual functional groups such as glyceride ester moieties and hydroxyl groups. At low RH, water uptake increased linearly with monoglyceride concentration while cooperativity was evident in water uptake profiles at high RH. Conclusions This study provides a potential universal framework for predicting relative drug solubility in mixtures containing fully saturated triglycerides and monoglycerides.     

Structures of Nanoparticles Prepared from Oil-in-Water Emulsions

Hydrophobic substances were dissolved in an organic solvent and emulsified with an aqueous solution at very high shear. Droplets of very small sizes (50–100 nm) were obtained by using surfactants which were combinations of lecithins and bile salts. After emulsification, the organic solvent was removed by evaporation, yielding stable dispersions of solid particles. The sizes, shapes, and structures of the particles were examined through quasi-elastic light scattering, small-angle neutron scattering and cryotransmission electron microscopy. Cholesteryl acetate particles stabilized by lecithin and bile salts were found to be platelets of 10–20 nm thickness and 80 nm diameter. Cholesteryl acetate particles stabilized with POE-(20)-sorbitan monolaurate were dense spherical globules of diameter 100 nm. Particles with a composition similar to the endogenously occurring lipoprotein, LDL, were large spherical globules studded with small vesicles. The subsequent evolution of the Cholesteryl acetate dispersion upon aging was examined. There was no transfer of cholesteryl acetate between particles nor to large crystals. However, some aggregation of the particles was observed when the volume fraction of the particles in the aqueous dispersion exceeded 0.05. Thus, the structure of the nanoparticles obtained through deswelling of emulsion droplets changes according to the nature of the emulsifiers and to the composition of the hydrophobic substances which they contain.     

Preparation and Evaluation of Multiple Emulsions Water-in-oil-in-water (w/o/w) as Delivery System for Influenza Virus Antigens

In this study, investigations have been carried out to prepare adjuvant active delivery systems; multiple water-in-oil-in-water (w/o/w) emulsion formulations, containing influenza virus surface antigen Hemagglutinin (HA). A modified two-stage emulsification method has been used to prepare multiple emulsions. After improving multiple (w/o/w) emulsion formulations; F1: purified antigen solution (PAS)/soybean oil, HCO-40 and span 80/pluronic F-68, F2: PAS and HPβCD/soybean oil, HCO-40 and span 80/pluronic F-68, F3: PAS/squalane, HCO-40 and span 80/pluronic F-68, formulations were selected for the stability study that continued for a 3 month duration. To evaluate the stability of these formulations, microscopic observation, osmolarities of the internal and external aqueous phases, pH, globule size and viscosity were determined. SDS-PAGE (silver staining) was used to evaluate HA and the micro-bicinchoninic acid (mBCA) assay was used to determine the in vitro release of antigen from formulations. Immune responses of formulations were investigated in Wistar Albino rats and compared with the immune response raised against the conventional vaccine. These responses were detected with Hemagglutination Inhibition (HAI) assay.

The results of this study demonstrated that HA was well entrapped in the multiple (w/o/w) emulsion formulations. Molecular weight and antigenicity of the entrapped HA were not affected by the emulsification procedure. These results suggest that multiple emulsion formulations entrapping influenza antigen may have potential for immunization studies as one of the vaccine delivery system with adjuvant properties.     

Analytically monitored digestion of silver nanoparticles and their toxicity on human intestinal cells

Orally ingested nanoparticles may overcome the gastrointestinal barrier, reach the circulatory system, be distributed in the organism and cause adverse health effects. However, ingested nanoparticles have to pass through different physicochemical environments, which may alter their properties before they reach the intestinal cells. In this study, silver nanoparticles are characterised physicochemically during the course of artificial digestion to simulate the biochemical processes occurring during digestion. Their cytotoxicity on intestinal cells was investigated using the Caco-2 cell model. Using field-flow fractionation combined with dynamic light scattering and small-angle X-ray scattering, the authors found that particles only partially aggregate as a result of the digestive process. Cell viabilities were determined by means of CellTiter-Blue® assay, 4′,6-diamidino-2-phenylindole-staining and real-time impedance. These measurements reveal small differences between digested and undigested particles (1–100 µg/ml or 1–69 particles/cell). The findings suggest that silver nanoparticles may indeed overcome the gastrointestinal juices in their particulate form without forming large quantities of aggregates. Consequently, the authors presume that the particles can reach the intestinal epithelial cells after ingestion with only a slight reduction in their cytotoxic potential. The study indicates that it is important to determine the impact of body fluids on the nanoparticles of interest to provide a reliable interpretation of their nano-specific cytotoxicity testing in vivo and in vitro.     

二甲酚橙共振光散射法测定DNA

用共振光散射技术,建立了以二甲酚橙为探针分子测定DNA的方法.在pH 3.5～4.3范围内,二甲酚橙在374nm处的共振光散射强度增强,且与DNA浓度呈线性关系,线性范围为2.5～15 μg/ml,检测限10ng/ml.     

激光散射法测定红参微粉的粒度分布

目的 建立激光散射法测定红参微粉粒径分布的方法。方法 采用Malvern Mastersizer 3000E激光粒度分析仪,AERO M型干法进样器进行测定。进样空隙高度：1.5 mm,分散气压：3.5 bar,进样速度：60%,折射率：1.52,样品和背景测量时间：30 s,吸收度：0.1,遮光度：0.5%~5%。结果 5批红参微粉重复性测定结果中,Dv(10)、Dv(50)、Dv(90)的RSD均<4%,符合中国药典2015年版相关要求。结论 激光散射测定法稳定可靠,可用于红参微粉的粒度分布测定。     

Release of 5-fluorouracil from intramuscular w/o/w multiple emulsions

Comparative in vivo studies of aqueous solution, multiple w/o/w, and w/o emulsions showed that formulating 5-fluorouracil in emulsion systems significantly sustained the release of the drug from intramuscular injection sites in the rat. Intramuscular injection of the drug in both w/o and w/o/w emulsion systems produced sustained blood concentrations with a later blood level peak than observed following intramuscular injection of aqueous solutions of the drug. The multiple w/o/w emulsion exhibited a more rapid release of drug from the injection site than the w/o emulsion because of partitioning of the drug to the external aqueous phase during secondary emulsification. The fate of the oil phase following intramuscular injection of a water/hexadecane/water multiple emulsion spiked with 1-14C-hexadecane has been studied in rats as a function of stabilizer concentrations. Increasing the lipophilic surfactant (Span 80) concentration facilitated the clearance of the oily vehicle from the injection site, by mechanisms which remain to be elucidated.     

激光散射法测定替格瑞洛原料药的粒度分布

目的：建立激光散射法测定替格瑞洛原料药的粒度分布。方法：采用 Malvern Mastersizer 2000激光粒度分析仪;Hydro 2000MU湿法进样器。样品折射率：1.52;样品吸光率：0.1;分散介质(水)折射率：1.33;泵速：1800rpm/min;超声频率：10;测量次数：3次;测量时间：5S;背景时间：5S;遮光度：10%-20%。结果：方法学考察结果d(0.5)的RSD＜2%,d(0.1)和d(0.9)的RSD均＜2%,3批替格瑞洛中试样品的d(0.1)≤2μm,d(0.5)≤5μm及d(0.9)≤15μm,符合《中国药典》的要求。结论：该方法操作简便、准确度高、耐用性好,适合替格瑞洛原料药的粒度检测。     

共振光散射光谱法测定依地酸钙钠注射液的含量

目的 建立一种快速测定依地酸钙钠注射液含量的方法。方法 采用共振光散射光谱技术测定0.01 mol·L^–1 NaOH的碱性条件下依地酸钙钠与草酸钠反应前后的共振光散射光谱强度差。结果 采用激发和发射光带宽5.0 nm、扫描速度2 000 nm·min^–1时,依地酸钙钠所解离的钙离子与草酸根形成草酸钙分子,从而使体系的共振光散射光谱强度减弱,在激发光与发射光波长均在273 nm处时,体系的共振光散射光谱强度差值最大,并在0.30~1.20 mg·L^–1内呈线性关系,相关系数R=0.999 2。本法检测限为0.005 8 mg·L^–1,定量限为0.019 3 mg·L^–1。结论 本法灵敏度高、专属性强、省时简便,可用于依地酸钙钠注射液的含量分析,测定值与容量分析法基本一致。     

激光散射法测定磺胺嘧啶银原料粒度分布

目的：建立激光散射法测定磺胺嘧啶银原料粒度分布。方法：采用Malvern Mastersizer 2000激光粒度分析仪;Hydro 2000 MU湿法进样器;泵速：2000 rpm ,样品折射率：1.679,颗粒吸收率：0.01,分散介质折射率：1.33,测量次数：5次;样品与背景测量时间：10 s,遮光度：10%～20%。结果：7批原料d（0.5）均小于10μm,RSD均小于6%,d（0.1）和d（0.9）均小于3.5μm和25μm, RSD均小于10%,符合《中国药典》相关要求。结论：所建立方法经考察,可用于磺胺嘧啶银原料的粒度测定。     

Effects of polymer,organic solvent and mixing strength on integrity of proteins and liposomes encapsulated in polymeric microspheres fabricated by the double emulsion process

The double emulsion process has commonly been applied to encapsulate water-soluble bioactive agents into polymeric microspheres. However, the integrity of many of these agents may be destroyed by the highly energetic procedures such as sonication that are routinely used to produce stable water-in-oil (w/o) emulsion. The aim of this research was to pursue the possibility of replacing the sonication by a mild emulsification procedure such as vortex mixing, with the use of certain materials to help to obtain stable w/o emulsion. The following materials were examined: poly(lactide-co-ethylene glycol) (PELA) as the polymer, ethyl acetate and acetone as the solvents, poly(vinyl alcohol) (PVA) and d-α tocopheryl polyethylene glycol 1000 succinate (Vitamin E TPGS) as the emulsifiers in w/o emulsion. The experimental results, with human serum albumin (HSA) as the encapsulated agent, showed that, when vortex mixing was used, these materials could significantly improve w/o emulsion stability and help to obtain satisfactory encapsulation effects, i.e. high encapsulation efficiency (EE) and low initial release burst. A delicate structure, i.e. liposomes, which is very sensitive to sonication, was then incorporated into microspheres by the ‘modified double emulsion process’. It was found that the liposomes were intact and the encapsulation effects were good. Therefore, it can be concluded that the modified double emulsion process could be advantageous for the encapsulation of delicate substances.     

激光散射法测定酪酸梭菌肠球菌三联活菌散粒度的探讨

摘 要 目的：建立测定酪酸梭菌肠球菌三联活菌散粒度分布的激光散射法,并与筛分法进行比较。方法: 激光散射法测试条件为：振动进样速度为80%,分散气压为0.05 MPa,背景及样品的扫描时间为15 s,遮光度为0.5%～5%,颗粒折射率为1.55,颗粒吸收率为0.01,进样量为0.1～0.2 g。测定粒度分布的特征值粒子体积累计分布图中10%、50%、90%处的粒径值[d（0.1）、d（0.5）、d（0.9）]和体积平均粒径D[4,3]。结果: 方法学考察中d（0.1）、d（0.5）、d（0.9）的RSD均小于5%。激光散射法结果显示93.3%的样品粒径小于250 μm,64.2%小于150 μm,51.4%小于125 μm,31.3%小于90 μm;筛分法结果显示96.6%的样品粒径小于250 μm,46.4%小于150 μm ,23.5%小于125 μm,1.4%小于90 μm。结论:筛分法和激光散射法均可以表征样品的粒度分布,但是激光散射法较筛分法的优点是样品量少、重复性好、测量速度快、测量范围广,可以全面表征样品的粒度分布。     

HPLC法测定蛇床子素的油/水分配系数

目的采用HPLC法研究了蛇床子素的油/水分配系数。方法通过测定已知logP值的系列化合物和蛇床子素的logK′值,来推算蛇床子素的logP值。结果采用C18,以乙腈-甲醇-磷酸氢二钠(11∶62∶27,0.05mol/L磷酸调pH值3.7)为流动相,检测波长254nm,流速1mL/min测定蛇床子素logP值为3.46。结论采用高效液相色谱法测定蛇床子素logP值具有快速、重现性好、样品不需特殊纯化及能测溶解度较小的化合物。     

Attenuation of acute and chronic restraint stress-induced perturbations in experimental animals by Zingiber officinale Roscoe

Ethanolic extract of rhizomes of Zingiber officinale was investigated on anoxia stress tolerance test in Swiss mice. The animals were also subjected to acute physical stress (swimming endurance test) to gauge the anti-stress potential of the extract. Further to evaluate the anti-stress activity of Z. officinale in chronic stress condition, fresh Wistar rats were subjected to cold restraint stress (4° for 2 h) for 10 days. Stimulation of hypothalamus pituitary adrenal axis in stressful condition alters plasma glucose, triglyceride, cholesterol, BUN and corticosterone levels. There is also alteration in the blood cell counts. Pretreatment with the extract significantly ameliorated the stress-induced variations in these biochemical levels and blood cell counts in both acute and chronic stress models. The extract treated animals showed increase in swimming endurance time and increase in anoxia tolerance time in physical and anoxia stress models, respectively. Treatment groups also reverted back increase in liver, adrenal gland weights and atrophy of spleen caused by cold chronic stress and swimming endurance stress models. The results indicate that ethanolic extract of Z. officinale has significant adaptogenic activity against a variety of biochemical and physiological perturbations in different stress models.     

Pharmacokinetics of Vancomycin after Intravenous Administration of a W/O/W Emulsion to Rats

A stable water-in-oil-in-water multiple emulsion (w/o/w emulsion) was prepared, and its potential for drug delivery was evaluated. W/o/w emulsions were prepared using a Lipiodol Ultra-Fluid(r) and isopropyl myristate oil mixture for the oily phase and vancomycin (VCM) for the entrapped drug. The surfactants, HCO-40 (5% [w/v]) and Pluronic F-88 were dissolved in the oily phase and the external aqueous phase, respectively. Resultant w/o/w emulsions were evaluated for entrapment efficiency, particle size, viscosity, drug release in vitro, and disposition kinetics of the drug and the w/o/w emulsion in vivo. The particle size of the w/o/w emulsion decreased with an increase in the concentration of F-88 in the external aqueous phase and stirring speed at the second emulsification stage (the smallest being 2.9 ± 1.5 μm). Entrapment efficiency of VCM in the w/o/w emulsion decreased with an increase in the concentration of F-88 (the maximum being 65.3 ± 5.4%). VCM release from the w/o/w emulsion was prolonged and tended to be slower with an increase in the particle size of the emulsion. After intravenous administration, significant differences in pharmacokinetic parameters, such as k₂₁, k_elβ, AUC₀-±, MRT₀-₆, and MRT₀-±, were observed between the VCM solution and the w/o/w emulsion-entrapped drug. When the w/o/w emulsion with Sudan II in the oily phase was administered intravenously, the emulsion accumulated in the lung at first (the highest value was observed just after administration) and then in the liver (the highest value was observed at 60 min). The w/o/w emulsion prepared in this study is expected to be a possible carrier for the prolonged release of water-soluble drugs after intravenous administration.     

High-Throughput In-Use and Stress Size Stability Screening of Protein Therapeutics Using Algorithm-Driven Dynamic Light Scattering

Stability of therapeutic proteins (TPs) is a critical quality attribute that impacts both safety and efficacy of the drug. Size stability is routinely performed during and after biomanufacturing. Dynamic light scattering (DLS) is a commonly used technique to characterize hydrodynamic size of the TPs. Herein, we have developed a novel method to evaluate in-use and thermal stress stability of TPs using algorithm-driven high-throughput DLS. Five marketed TPs were tested under the guidance of customized algorithms. The TPs were evaluated at relevant temperature conditions as well as under dilution and thermal stress for size stability. We found that the TPs were stable under the in-use conditions tested; however, sample loss due to evaporation can lead to large protein aggregates. A combined assessment of autocorrelation function and photos of sample well could be useful in formulation screening. Dilution of TPs also has an impact on the hydrodynamic size. Thermal stress experiments showed the importance of using different data processing methods to access size distribution. Polydispersity index was useful in evaluating sample heterogeneity. Herein, we show that algorithm-driven high-throughput DLS can provide additional supportive information during and after biomanufacturing and the potential to be used in a quality control environment.     

磺胺二甲嘧啶的溶解度及油水分配系数的研究

目的:研究磺胺二甲嘧啶(SM2)在不同pH值下的平衡溶解度和油水分配系数(Ko/w),为SM2制剂的开发提供基础研究。方法:配制不同pH值的磷酸盐缓冲液,采用饱和法测定其表观溶解度;通过SM2分配平衡后在油相(正辛醇)和水相的浓度比,计算油水分配系数。结果:SM2在pH2·0和pH9·0时平衡溶解度较高,分别为1·916、1·375g·L-1,油水分配系数在pH8·0时最高,为3·9070。结论:SM2在水中的平衡溶解度及油水分配系数与介质的pH值有关,pH<3·0或pH>6·8时溶解度较好。pH3～8时SM2在油相中分配较多,较易被机体吸收。     

Rapid and soft formulation of folate-functionalized nanoparticles for the targeted delivery of tripentone in ovarian carcinoma

We report the development of folate-functionalized nanoparticles able to target folate receptors, and to deliver a poorly water soluble cytotoxic agent, a tripentone, in ovarian carcinoma. The stability under incubation of lipid nanoparticles formulated by a low-energy phase inversion temperature method was investigated. Thanks to the presence of Labrasol^®, a macrogolglyceride into the composition of the nanocarriers, the conjugation of different quantities of a folate derivate (folic acid-polyethylene glycol₂₀₀₀-distearylphosphatidylethanolamine) to nanoparticles was possible by a rapid, soft, very simple post-insertion process. As determined by dynamic light scattering, nanoparticles present a monodisperse diameter of about 100 nm, a spherical shape as attested by transmission electron micrographs, a weakly negative surface zeta potential, and are able to encapsulate the tripentone MR22388. The presence of folate receptors on SKOV3 human ovarian cancer cells was identified by fluorescent immunocytochemistry. Cellular uptake studies assessed by flow cytometry indicated that these nanoparticles reached the SKOV3 cells rapidly, and were internalized by a folate-receptor mediated endocytosis pathway. Moreover, nanoparticles allowed the rapid delivery of the antitumor agent tripentone into cells as shown in vitro by real-time cellular activity assay. Such folate-lipid nanoparticles are a potential carrier for targeted delivery of poorly water soluble compounds into ovarian carcinoma.     

Aggregation of Aβ Alzheimer's disease-related peptide studied by dynamic light scattering

The aggregation behavior of the major component of Alzheimer's disease-related, amyloid peptides, Aβ-(1–40) and Aβ-(1–42), was studied in solution using dynamic light scattering. With most solvents employed, we found fibrils coexisting with oligomeric Aβ species. Pronounced differences were observed in aggregation of Aβ-(1–40) and (1–42) sequences in acetonitrile-water mixtures. Cofactors such as Zn²⁺ were found to induce deaggregation of Aβ instead of aggregation. The results indicated that the initial state of the peptide immediately after synthesis is rather poorly defined. Using freezing instead of lyophilization after the final peptide synthesis step, may partially relieve these problems.