Ethanol-based proliposome delivery systems of paclitaxel for in vitro application against brain cancer cells

In this study the anticancer activity of paclitaxel-loaded nano-liposomes on glioma cell lines was investigated. Soya phosphatidylcholine:cholesterol (SPC:Chol), hydrogenated soya phosphatidylcholine:cholesterol (HSPC:Chol) or dipalmitoylphosphatidylcholine:cholesterol (DPPC:Chol) in 1:1?mole ratio were used to prepare ethanol-based proliposomes. Following hydration of proliposomes, the size of resulting vesicles was subsequently reduced to nanometer scale via probe-sonication. The resulting formulations were characterized in terms of size, zeta potential and morphology of the vesicles, and entrapment efficiency of paclitaxel (PX) as well as the final pH of the preparations. DPPC-liposomes entrapped 35–92% of PX compared to 27–74% and 25–60% entrapped by liposomes made from SPC and HSPC formulations respectively, depending on drug concentration. The entrapment efficiency of liposomes was dependent on the lipid bilayer properties and ability of PX to modify surface charge of the vesicles. In vitro cytotoxicity studies revealed that PX-liposome formulations were more selective at inhibiting the malignant cells. The cytotoxicity of PX-liposomes was dependent on their drug-entrapment efficiency. This study has shown PX-liposomes generated from proliposomes have selective activity against glioma cell lines, and the synthetic DPPC phospholipid was most suitable for maximized drug entrapment and highest activity against the malignant cells in vitro.     

Preparation and characterization of iron-containing liposomes: their effect on soluble iron uptake by Caco-2 cells

The aim of this work was to study the iron uptake of Caco-2 cells incubated with five different formulations of liposomes containing iron. The vesicles were also characterized before, during, and after in vitro digestion. Caco-2 cells were incubated with digested and nondigested liposomes, and soluble iron uptake was determined. Nondigested liposomes made with chitosan (CHI) or the cationic lipid, DC-Cholesterol (DC-CHOL), generated the highest iron uptake. However, these two formulations were highly unstable under in vitro digestion, resulting in nonmeasurable iron uptake. Digested conventional liposomes composed of soybean phosphatidylcholine (SPC), hydrogentated phosphatidylcholine (HSPC), or HSPC and cholesterol (CHOL) presented the highest iron-uptake values. These liposomal formulations protected iron from oxidation and improved iron uptake from intestinal cells, compared to an aqueous solution of ferrous sulphate.     

Physicochemical study of the protein–liposome interactions: influence of liposome composition and concentration on protein binding

Abstract

The aim of the present study is to investigate the interactions between liposomes and proteins and to evaluate the role of liposomal lipid composition and concentration in the formation of protein corona. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or hydrogenated soybean phosphatidylcholine (HSPC) with 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (DPPE-PEG 3000), cholesterol (CH) or mixtures of these lipids, were prepared at different concentrations by the thin-film hydration method. After liposomes were dispersed in HPLC-grade water and foetal bovine serum (FBS), their physicochemical characteristics, such as size, size distribution, and ζ-potential, were determined using dynamic and electrophoretic light scattering. Aggregation of DPPC, HSPC, DPPC:CH (9:1 molar ratio), and HSPC:CH (9:1 molar ratio) in FBS was observed. On the contrary, liposomes incorporating DPPG lipids and CH both in a molar ratio of 11% were found to be stable over time, while their size did not alter dramatically in biological medium. Liposomes containing CH and PEGylated lipids retain their size in the presence of serum as well as their physical stability. In addition, our results indicate that the protein binding depends on the presence of polyethylene glycol (PEG), CH, concentration and surface charge. In this paper, we introduce a new parameter, fraction of stealthiness (F_s), for investigating the extent of protein binding to liposomes. This parameter depends on the changes in size of liposomes after serum incubation, while liposomes have stealth properties when F_s is close to 1. Thus, we conclude that lipid composition and concentration affect the adsorption of proteins and the liposomal stabilization.     

Quantification of various phosphatidylcholines in liposomes by enzymatic assay

The purpose of this research was to adapt a colorimetric, phospholipase D-based serum-phospholipid assay for the quantification of phosphatidylcholine (PC) in liposomes using a microtitre plate reader. PC from natural egg PC liposomes was quantified reliably. In contrast, poor sensitivity was found for liposomes composed of saturated PCs (dipalmitoyl-phosphatidylcholine [DPPC], hydrogenated egg PC). Triton X-100 was then added to the liposomes followed by heating above the phase transition temperature. This modified sample preparation resulted in recoveries of 102.6%±1.0%, 104.4%±7.6%, and 109.4%±3.2% for E80, E80-3/cholesterol, and DPPC liposomes, respectively. Absolute quantification of unknown PCs against a choline chloride standard is feasible, but relative measurements against the very same PC are recommended wheneve possible. Validation experiments revealed an absolute quantification limit of 1.25 μg per assay, a good linearity in the range of 25 to 1000μg/mL PC (r²≥0.9990) and a quite high accuracy (99.8%–101.4% of theory) and precision (relative standard deviation ≤3.2%) for all 3 PCs studied. The method is thus regarded as suitable for sensitive, rapid, and reliable routine quantification of PCs in liposomes.     

Lipid Dependent Cardio- Haemodynamic Tolerability of Liposomes in Rats

Abstract

Rationale and Objectives:

The use of contrast-carrying liposomes in diagnostic applications (1) or of haemoglobin liposomes in blood replacement therapy (2) requires infusion of large lipid doses. Saturated lipids like HSPC are often used in these formulations to render the liposomes more stable (3). Previous studies have indicated that intravenous injection of such liposome preparations can result in significant haemodynamic changes in rats (14). The purpose of this study was to systematically evaluate cardio- and haemodynamic effects of liposomes prepared from saturated and unsaturated phosphatidylcholine alone or in combination with other lipid components.

Methods;

Liposomes made from SPC, HSPC, DSPC, DSPC/CH, DSPC/DSPG, DSPC/CH/DSPG were infused in anaesthetized rats (total lipid dose: 300 mg lipid/kg BW) and cardio-heamodynamic parameters were measured.

Results:

DSPC-liposomes significantly reduced blood pressure (BP) and total peripheral resistance (TPR) by -53.7 % and -45.7 % of prevalue, respectively. Similar results were obtained for HSPC-liposomes. Marked ECG-changes were recorded for both formulations. SPC-liposomes caused a transient and moderate reduction of BP and TPR (-17.0 % and -22.3 %, respectively). Short-lasting ECG changes were also observed. The addition of cholesterol or DSPG to DSPC liposomes reduced cardiac and haemodynamic side effects in rats.

Conclusion;

The lipid composition of liposomes is of major importance for the incidence of cardiovascular side effects in rats. Liposomes composed of pure saturated phosphatidylcholine cause significant changes which can be diminished by the addition of other lipid components like cholesterol.     

Combined strategies for liposome characterization during in vitro digestion

Three types of pyranine (HPTS)-containing liposomes were prepared by high-pressure homogenization under optimized conditions. At 37°C, they were 1) fluid-state vesicles made from soybean phosphatidylcholine (SPC), 2) gel-state liposomes made from hydrogenated SPC (HSPC), and 3) solid-disordered membranes obtained from HSPC and cholesterol (HSPC-Chol). These liposome formulations were characterized before, during, and after in vitro digestion, which involved the presence of pH gradients, enzymes, and bile salts. Mean sizes and size distributions of the vesicles were determined by DLS; ³¹P-NMR (nuclear magnetic resonance) was used to quantify lyso-PC forms; internal pH was monitored throughout digestion with two different fluorescent pH probes; and changes in bilayer permeability and HPTS encapsulation were determined by size-exclusion chromatography and fluorimetry. Differential scanning calorimetry analysis was also performed in order to study the effect of digestion on HSPC vesicles. SPC liposomes were physically stable during digestion; they presented 8% lyso-forms and an HPTS encapsulation around 85% after in vitro digestion. However, they were extremely permeable to ions, so that the internal pH immediately equilibrated with the bulk pH. HSPC liposomes were the most affected by the digestive process. Even though they were chemically stable, as inferred from the low lyso-PC content, very important changes in their size distribution were observed. A final 50% HPTS leakage was quantified after in vitro digestion. Nevertheless, they were the least permeable to protons under pH gradients. HSPC-Chol vesicles presented intermediate permeability to protons, having their internal pH decreased from approximately 6.8 to 4.6 after 1 hour of incubation at pH 2. This was the most chemically stable formulation and showed the highest encapsulation, even after in vitro digestion. Therefore, HSPC-Chol liposomes would be the most adequate choice for the design of lipid products for oral administration.     

Freeze-drying of ATP entrapped in cationic,low lipid liposomes

Concerning the instability of ATP liposomes formulated to easily diffuse through the liver (size ～100 nm), this work targets the key parameters that influence the freeze-drying of a preparation that combines cholesterol, DOTAP and phosphatidylcholine (either natural soybean or egg (SPC or EPC) or hydrogenated (HSPC)). After freeze-drying blank liposomes, size increased significantly when initial lipid concentration was lowered from 20 to 5 mM (p = 0.0018). With low lipid concentration preparation (5 mM), SPC limited size increase (SI) more efficiently compared to EPC or HSPC. With SPC and EPC, sucrose showed better size results compared to trehalose (Lyoprotectant/Lipid ratio (w/w) avoiding any SI: ～5 and ～10 (for SPC), ～10 and ～15 (for EPC), for sucrose and trehalose, respectively), but the opposite was evidenced with HSPC liposomes where a Trehalose/Lipid ratio of 25 barely prevented SI. In addition, slow versus quick cooling rate led to limiting SI for HSPC liposomes (p = 0.0035). With sucrose or trehalose at both Lyoprotectant/Lipid ratios ensuring size stabilisation (10:1 and 15:1, respectively), ATP leakage ranged between 38.8 ± 7.9% and 58.2 ± 1.4%. In conclusion, this study emphasizes that using strict size maintenance as the primary objective does not result in drug complete retention inside the liposome core.     

Mechanical grinding effect on thermodynamics and inclusion efficiency of loratadine-cyclodextrin inclusion complex formation

The interaction of poor water-soluble drug loratadine (LOR) with β-cyclodextrin (β-CD) or hydroxypropyl-β-cyclodextrin (HP-β-CD) in aqueous or solid state was investigated. Mechanical grinding effect on the inclusion steps, thermodynamic kinetics and inclusion efficiency of inclusion complex formation of LOR with β-CD or HP-β-CD was quantitatively investigated by DSC and FT-IR microspectroscopy with curve-fitting analysis. The phase solubility profiles of LOR with β-CD and HP-β-CD were classified as A_L-type phase diagram. The grinding-induced reduction in LOR crystallinity in the presence of β-CD or HP-β-CD was found to be apparent zero-order kinetics. The inclusion efficiency of solid inclusion complex for LOR/β-CD or LOR/HP-β-CD was significantly correlated with the reduction in LOR crystallinity and the grinding time. The mechanism of inclusion complex formation for LOR/β-CD or LOR/HP-β-CD was proposed through the progressive reduction in LOR crystallinity, the promoted LOR amorphization, and molecular inclusion processes in the continuous energy input process of mechanical grinding.     

Quantification of trace levels of β-cyclodextrin and substitution patterns in hydroxypropyl-β-cyclodextrin using high-performance liquid chromatography with polarimetric detection

A high-performance liquid chromatographic (HPLC) method has been developed for separation and determination of components in hydroxypropyl-β-cyclodextrin (HP-β-CD). The method involves separation on an amino-bonded HPLC column using water–acetonitrile as a mobile phase with a polarimetric HPLC detector for quantification. It provides good selectivity and sensitivity and can also be used to compare different sources of HP-β-CD and to measure batch to batch variation. The similarity of the values of molar optical rotation for β-cyclodextrin (β-CD) and HP-β-CD suggests that a polarimetric HPLC detector may be used with a straightforward area normalization method, to quantify the proportion of β-CD in any HP-β-CD sample. Trace amounts of β-CD in HP-β-CD have been measured to a precision of 0.01%. © 1993 Wiley-Liss, Inc.     

Phospholipid dependence of calcium ion effects on electrophoretic mobilities of liposomes

Electrophoretic light scattering (ELS) and depolarization of fluorescence have been used to determine the effect of membrane fluidity on the binding of Ca2+ to liposomes. ELS was used to measure the electrophoretic mobilities of the liposomes. Fluorescence depolarization was used to determine membrane fluidity. Zero to 30 mol% phosphatidylserine (PS) was incorporated into liposomes containing, as bulk phospholipids, one of the following: dimyristoyl-phosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (PC), or hydrogenated egg phosphatidylcholine (H egg PC). The binding of Ca2+ to the liposomes appears to be influenced by membrane fluidity. Liposomes containing bulk phospholipids whose phase transition temperature is higher than the experimental temperature exhibit enhanced binding of CA2+.     

Micellar formation of spin-labeled fatty acyl derivatives of lipophilic muramyl dipeptides and their incorporation into liposomal membranes

A lipophilic muramyl dipeptide (MDP) with a nitroxide moiety in its acyl chain (SL-MDP) and its N-methyl derivative (SL-methyl MDP) were synthesized. The SL-MDPs formed micelles (cmc, 0.1-0.3 mM). The ESR spectra of the SL-MDPs in phosphatidylcholine (PC) liposomes at 25 degrees C consisted of an anisotropic signal and three sharp lines, indicating that both SL-MDPs partitioned between membranes and aqueous phase. The amounts of the SL-MDPs in membranes depended on the phospholipid species and the cholesterol (Chol) content, but no appreciable difference was observed between SL-MDPs. The SL-MDPs partitioned well at 25 degrees C into egg yolk PC liposomes but not into pure dipalmitoylphosphatidylcholine (DPPC), suggesting that the incorporation may be related to the membrane fluidity. Chol enhanced the incorporation into both phospholipids. The mobilities of the SL-MDPs in the membranes were less than that of the corresponding spin-labeled fatty acid. Comparison of the mobilities among SL-MDPs, spin-labeled ganglioside and spin-labeled galactosylceramide showed that the hydrophilicity of the polar group may influence the immobilization of their acyl chains.     

Investigation of inclusion complex of cilnidipine with hydroxypropyl-β-cyclodextrin

The objective of this study was to improve the water-solubility and photostability of cilnidipine by complexing it with hydroxypropyl-β-cyclodextrin (HP-β-CD or HP-beta-CD). The interactions of cilnidipine and HP-β-CD were characterized by ultra violet-visible (UV/VIS) spectroscopy, differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), Fourier transformation-infrared (FT-IR) spectroscopy and (1)H nuclear magnetic resonance ((1)H NMR) spectroscopy to verify the formation of cilnidipine-HP-β-CD complex inclusion. Moreover, the binding sites in the HP-β-CD structure were also tracked through (1)H NMR spectroscopy analysis. All the characterization information proved the formation of cilnidipine-HP-β-CD inclusion complex, and the results demonstrated the superiority of the inclusion complex in dissolution rates and photostability; in addition, the apparent solubility of cilnidipine was increased more than 10,000-fold in the presence of HP-β-CD. The stability constant (1:1) was found to be 50,116M(-1), suggesting a high tendency of the drug to enter the HP-β-CD cavity. These results identified the cilnidipine-HP-β-CD inclusion complex as an effective new approach to design a novel formulation for pharmaceutical application.     

Cleaning efficacy of hydroxypropyl-beta-cyclodextrin for biofouling reduction on reverse osmosis membranes

In this study, an environmentally friendly compound, hydroxypropyl-beta-cyclodextrin (HP-β-CD) was applied to clean reverse osmosis (RO) membranes fouled by microorganisms. The cleaning with HP-β-CD removed the biofilm and resulted in a flux recovery ratio (FRR) of 102%. As cleaning efficiency is sometimes difficult to determine using flux recovery data alone, attached bacterial cells and extracellular polymeric substances (EPS) were quantified after cleaning the biofouled membrane with HP-β-CD. Membrane surface characterization using scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared (ATR-FTIR) and atomic force microscopy (AFM) confirmed the effectiveness of HP-β-CD in removal of biofilm from the RO membrane surface. Finally, a comparative study was performed to investigate the competitiveness of HP-β-CD with other known cleaning agents such as sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), Tween 20, rhamnolipid, nisin, and surfactin. In all cases, HP-β-CD was superior.     

Stability of small unilamellar liposomes in serum and clearance from the circulation: The effect of the phospholipid and cholesterol components

Small unilamellar liposomes containing carboxyfluorescein (CF) and composed of various unsaturated and saturated phospholipids with or without cholesterol were incubated in the presence of mouse serum at 37°C. Liposomes composed of egg L-α-phosphatidylcholine (PC), L-α-dioleoylphosphatidylcholine (DOPC) or sphingomyelin (SM) became rapidly permeable to entrapped CF but incorporation of cholesterol into such liposomes reduced CF leakage. Under similar conditions, CF leakage from cholesterol-free liposomes composed of saturated phospholipids of increasing fatty acid chain length was dependant on the liquid-crystalline phase transition temperature (Tc) of the phospholipid component. Thus, L-α-dilaureoylphos-phatidylcholine (DLPC), L-α-dimyristoyl phosphatidylcholine (DMPC) and L-α-dipalmitoylphosphatidylcholine (DPPC) with Tc's below or near the temperature of the incubation (37°C) released CF rapidly whereas L-α-diheptedecanoyl phosphatidylcholine (DHPC), L-α-distearoylphosphatidylcholine (DSPC) and hydrogenated egg PC (HPC) liposomes with Tc's above 37°C retained the dye quantitatively. After incorporation of cholesterol into liposomes composed of saturated phospholipids, CF release was reduced for DLPC and DMPC and increased for DPPC, DSPC, DHPC and HPC vesicles. Liposomes with or without cholesterol exhibiting greatest stability (in terms of CF retention) in the presence of serum were injected intravenously into mice and rates of clearance of quenched CF from the circulation measured. Observed clearance rates were linear and, when liposomes contained tritiated phospholipid, identical to those of the radiolabel suggesting retention of liposomal integrity in the intravascular space. However, half-lifes of liposomes ranging from 0.1 to 16 h did not correlate with the physical characteristics of their phospholipid component. After intraperitoneal injection, there was quantitative entry of quenched CF (stable liposomes) into the blood from which it was eliminated at rates corresponding to those observed after intravenous injection. These results suggest that solute retention by liposomes and their half-life in the circulation can be controlled by the appropriate manipulation of liposomal membrane fluidity and composition.     

Direct enantioseparation of mandelic acid by high-performance liquid chromatography using a phenyl column precoated with a small amount of cyclodextrin additive in a mobile phase

Direct enantioseparation of mandelic acid by high-performance liquid chromatography (HPLC) with a reversed phase column and a mobile phase containing a small amount of hydroxylpropyl-β-cyclodextrin (HP-β-CD) was studied as an efficient method for saving consumption of the CD additive. As a result, it was proposed that racemic mandelic acid can be analyzed with a phenyl column by using a mobile phase composed of 10 mM ammonium acetate buffer (pH 4.2) and 0.02% (w/v) HP-β-CD at a flow rate of 1.0 mL/min at 40°C after the passage of 10 mM ammonium acetate buffer (pH 4.2) containing 0.1% (w/v) HP-β-CD as a precoating mobile phase for 60 min. It is suggested that HP-β-CD is bound with a phenyl group on the surface of the stationary phase to allow a phenyl column to act as a transient chiral column, and injected mandelic acid can form the ternary complex with the adsorbed HP-β-CD. The longer retention time of D-mandelic acid than the L-isomer for HPLC can be explained from the higher stability of the HP-β-CD complex with D-mandelic acid, which was confirmed by CE experiment with HP-β-CD as a selector. The efficiency of a phenyl column compared with other stationary phases was also discussed.     

Studies on trimethoprim:hydroxypropyl-β-cyclodextrin: aggregate and complex formation

The present study is focused on the characterization of the interaction between trimethoprim, a dihydropteroate synthesase inhibitor, and hydroxypropyl-β-cyclodextrin (HP-β-CD) in aqueous solution and solid state. The freeze-drying method was used to prepare solid complexes, while simple blending was employed to obtain physical mixtures. The phase solubility was AN type, and demonstrated that trimethoprim solubility was significantly increased upon complexation with HP-β-CD. Conductivity experiments showed the presence of aggregates that explains the type profile for the solubility isotherm. The critical concentration for the aggregate formation was determined to be 69.3 mg/ml for pure HP-β-CD and 117.7 mg/ml in the presence of trimethoprim. Nuclear magnetic resonance spectroscopy provided evidence of trimethoprim:HP-β-CD molecular interaction in solution. Moreover, the complex was characterized in solid stated using Fourier-transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). The use of differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) showed that the thermal stability of the drug is enhanced in the presence of HP-β-CD.     

Enhancement of rhubarb extract solubility and bioactivity by 2-hydroxypropyl-β-cyclodextrin

Rhubarb is a traditional Chinese medicinal herb, and the ethanolic extract of rhubarb consists of active anthraquinones, which are hydrophobic and have antiproliferative effects on hepatoma cell lines. To increase the aqueous solubility of rhubarb and study the consequent bioavailability, the ethanolic extract of rhubarb was complexed with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), a cyclic oligosaccharide that has a hydrophilic outer surface and a hydrophobic central cavity, to form a rhubarb–HP-β-CD complex. This complex was characterized by performing nuclear magnetic resonance spectroscopy, two-dimensional rotating frame spectroscopy and thin layer chromatography to confirm the inclusion of anthraquinones from rhubarb extract in HP-β-CD (weight ratio of rhubarb extract:HP-β-CD = 1:9). We investigated the effects of complexing rhubarb extract with HP-β-CD on the growth of Huh7 and HepG2 cells by performing cytotoxicity analysis, cellular uptake test, and colony formation assay. Our results showed that complexation of rhubarb extract with HP-β-CD increased the aqueous solubility and bioavailability of rhubarb and thus enhanced its effect on hepatoma cells.     

Spectroscopic and calorimetric studies on trazodone hydrochloride–phosphatidylcholine liposome interactions in the presence and absence of cholesterol

The interaction of antidepressant drug trazodone hydrochloride (TRZ) with dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) in the presence and absence of cholesterol (CHO) was investigated as a function of temperature by using Electron Paramagnetic Resonance (EPR) spin labeling, Fourier Transform Infrared (FTIR) Spectroscopy and Differential Scanning Calorimetry (DSC) techniques. These interactions were also examined for dimyristoyl phosphatidylcholine (DMPC) multilamellar liposomes by using Electron Paramagnetic Resonance (EPR) spin labeling technique. In the EPR spin labeling studies, 5- and 16-doxyl stearic acid (5-DS and 16-DS) spin labels were used to monitor the head group and alkyl chain region of phospholipids respectively. The results indicated that TRZ incorporation causes changes in the physical properties of PC liposomes by decreasing the main phase transition temperature, abolishing the pre-transition, broadening the phase transition profile, and disordering the system around the head group region. The interaction of TRZ with unilamellar (LUV) DPPC liposomes was also examined. The most pronounced effect of TRZ on DPPC LUVs was observed as the further decrease of main phase transition temperature in comparison with DPPC MLVs. The mentioned changes in lipid structure and dynamics caused by TRZ may modulate the biophysical activity of membrane associated receptors and in turn the pharmacological action of TRZ.     

Formulation and Development of Extended-Release Micro Particulate Drug Delivery System of Solubilized Rifaximin

Rifaximin (RFX), a semi-synthetic antibiotic belonging to BCS class IV category, has been used in the treatment of traveler’s diarrhea. An attempt has been made to improve aqueous solubility of RFX in the presence of β-cyclodextrin (β-CD) and hydroxy propyl β-cyclodextrin (HP-β-CD) and control its release in the gut by enteric coating. The stoichiometric proportion of RFX and complexing agent’s β-CD and HP-β-CD were determined by phase solubility studies. RFX–β-CD and RFX–HP-β-CD were prepared in 1:2 ratio by solvent evaporation technique using rota-evaporator with yield of 78% and 84% respectively followed by their evaluation using different techniques such as saturation solubility, Fourier transform infrared, differential scanning calorimeter, powder X-ray diffractometer, in vitro antimicrobial activity. The saturation solubility of RFX had improved from 0.0736 mg/ml to 0.2354 mg/ml and 0.5681 mg/ml in presence of β-CD and HP-β-CD respectively resulting in an increased zone of inhibition in the later complex during antimicrobial studies. The RFX–HP-β-CD complex particles were coated with eudragit L 100 (EL 100) by spray drying technique. The 3² factorial design was applied to formulate the micro particles. All formulations exhibited pH dependant drug release. The % EE was 69% and the release of RFX was retarded by enteric coating in the optimized batch FB2. Therefore, it can be concluded that solubility of some BCS class IV drugs can be improved by β-CD complexation and release of such inclusion complexes can be retarded to increase the residence time of RFX in the gastrointestinal tract.     

Incorporation and modification of exogenous phosphatidylcholines by mycoplasmas.

The uptake and modification of exogenous phosphatidylcholine (PC) by several Mycoplasma and Spiroplasma species was investigated. While in most Mycoplasma species and in all Spiroplasma species tested the PC appears to be incorporated unchanged from the growth medium, the PC of M. gallisepticum, M. pulmonis, and M. pneumoniae was disaturated PC, apparently formed by modification of 1-saturated-2-unsaturated PC from the growth medium. The modification of the exogenous PC by M. gallisepticum was inhibited by chloramphenicol under conditions that did not affect de novo synthesis of phosphatidylglycerol. A low activity of an endogenous phospholipase A was detected in native M. gallisepticum membranes. The activity was markedly stimulated by treating the membranes with low concentrations of the nonionic detergents. The PC modification was affected by the fatty acid composition of the exogenous PC species. Diunsaturated, 1-saturated-2-unsaturated, and 1-unsaturated-2-saturated PCs were modified to various extents, whereas the disaturated dipalmitoyl PC (DPPC) was not. Both modified and unmodified PCs were incorporated by the cells, but the unmodified DPPC was incorporated at a lower rate and to a lesser extent. The possibility that the incorporation of DPPC into M. gallisepticum cells is associated with the formation of intracytoplasmic membranes is discussed.