Cellular Vesicles: New Insights in Engineering Methods, Interaction with Cells and Potential for Brain Targeting.

Cellular vesicles (CVs) have been proposed as alternatives to exosomes for targeted drug delivery. CVs, prepared from human embryonic kidney 293 cells (HEK-293), C57BL/6 mouse B16F10 skin melanoma cells (B16F10), and immortalized human cerebral microvascular endothelial cells (hCMEC/D3) by liposome technology methods, were characterized for morphology, cytotoxicity, and cell uptake properties. CV brain-targeting potential was evaluated in vitro on the hCMEC/D3 blood-brain barrier (BBB) model, and in vivo/ex vivo. CV sizes were between 135 and 285 nm, and the ζ-potential was negative. The dehydration-rehydration method conferred highest calcein loading and latency to CVs compared with other methods. The increased calcein leakage from CVs when compared with liposomes indicated their poor integrity, which was increased by pegylation. The in vivo results confirmed lower liver uptake by PEG-CVs (compared with nonpegylated) proving that the calcein integrity test is useful for prediction of CV biodistribution, as used for liposomes. The cell uptake of homologous origin CVs was not always higher compared with that of non-homologous. Nevertheless, CVs from hCMEC/D3 demonstrated the highest BBB permeability (in vitro) compared with OX-26 targeted liposomes, and brain localization (in vivo). CVs from hCMEC/D3 cells grown in different media demonstrated decreased interaction with brain cells and brain localization. Significant differences in proteome of the two latter CV types were identified by proteomics, suggesting a potential methodology for identification of organotropism-determining CV components.

Mono and dually decorated nanoliposomes for brain targeting, in vitro and in vivo studies.

PURPOSE: Mono- and dual-decorated (DUAL) liposomes (LIP) were prepared, by immobilization of MAb against transferrin (TfR[OX26 or RI7217]) and/or a peptide analogue of ApoΕ3 (APOe) -to target low-density lipoprotein receptor(LPR)-, characterized physicochemically and investigated for BBB-targeting, in-vitro and in-vivo. METHODS: Human microvascular endothelial cells (hCMEC/D3) were used as BBB model, and brain targeting was studied by in-vivo imaging of DiR-labelled formulations (at two doses and surface ligand densities), followed by ex-vivo organ imaging. RESULTS: LIP diameter was between 100 nm and 150 nm, their stability was good and they were non-cytotoxic. LIP uptake and transport across the hCMEC/D3 cell monolayer was significantly affected by decoration with APOe or MAb, the DUAL exerting an additive effect. Intact vesicle-transcytosis was confirmed by equal transport of hydrophilic and lipophilic labels. In-vivo and ex-vivo results confirmed MAb and DUAL-LIP increased brain targeting compared to non-targeted PEG-LIPs, but not for APOe (also targeting ability of DUAL-LIP was not higher than MAb-LIP). The contradiction between in-vitro and in-vivo results was overruled when in-vitro studies (uptake and monolayer transport) were carried out in presence of serum proteins, revealing their important role in targeted-nanoformulation performance. CONCLUSIONS: A peptide analogue of ApoΕ3 was found to target BBB and increase the targeting potential of TfR-MAb decorated LIP, in-vitro, but not in-vivo, indicating that different types of ligands (small peptides and antibodies) are affected differently by in-vivo applying conditions. In-vitro tests, carried out in presence of serum proteins, may be a helpful predictive "targetability" tool.

Development of growth factor-incorporating liposomes for integration into scaffolds as a method to improve tissue regeneration.

This review is an update with regard to the efforts to develop liposomal carriers for growth factor delivery. It is well known that growth factors have the potential to enhance/accelerate tissue regeneration; however, their poor stability, which results in rapid loss of their activity, together with their rapid clearance from defected tissues (when applied as free molecules) is a serious drawback for their use; their highly hydrophilic nature and low capability to permeate through biological barriers (cell membranes) are additional factors that limit their applicability. In recent years, the advantages of liposomal drug delivery systems have motivated efforts to deliver growth factors (GFs) in liposomal form. Herein, after briefly introducing the basic structural characteristics of liposome types and their advantages when used as drug carriers, as well as the basic problems encountered when GFs are applied for tissue regeneration, we focus on recent reports on the development and potential regenerative effects of liposomal GFs, towards defects of various tissues. The methodologies used for incorporation, attachment or immobilization of liposomal GFs in order to sustain their retention at the defected tissues are also highlighted.

Overcoming barriers by local drug delivery with liposomes.

Localized or topical administration of drugs may be considered as a potential approach for overcoming the problems caused by the various biological barriers encountered in drug delivery. The combination of using localized administration routes and delivering drugs in nanoparticulate formulations, such as liposomes, may have additional advantages. Such advantages include prolonged retention of high drug loads at the site of action and controlled release of the drug, ensuring prolonged therapeutic effect; decreased potential for side-effects and toxicity (due to the high topical concentrations of drugs); and increased protection of drugs from possible harsh environments at the site of action. The use of targeted liposomal formulations may further potentiate any acquired therapeutic advantages. In this review we present the most advanced cases of localized delivery of liposomal formulations of drugs, which have been investigated pre-clinically and clinically in the last ten years, together with the reported therapeutic advantages, in each case.

Novel Liposome Aggregate Platform (LAP) system for sustained retention of drugs in the posterior ocular segment following intravitreal injection.

A novel Liposome Aggregate Platform (LAP) system for prolonged retention of drugs in the posterior eye segment after intravitreal injection (IVT) was developed and evaluated. Calcein, FITC-dextran-4000 (FD4) and Flurbiprofen (FLB), were encapsulated in negatively charged liposomes, and mixed with protamine to produce the LAP. The lipid/protamine ratio was fixed, in order to have a convenient aggregation rate permitting IVT injection and also a sustained release of liposome-entrapped molecules (in vitro) from LAP. In vitro release studies confirmed the potential of LAP system consisted of HPC/DPPG/Chol liposomes and protamine (at 1:1 w/w to lipid), to delay calcein, FD4 and FLB release, compared to free liposomes. In vivo studies demonstrated increased vitreous retention of liposomes and LAP for all molecules, compared to the corresponding solutions; however the retention of FD4 is similar for non-aggregated liposomes and LAP, and calcein retention is only slightly increased by LAP compared to liposomes. The later result may be connected with the visible ocular inflammation caused by both dyes; interestingly inflammation was moderately reduced when dyes were entrapped in liposomes and even more when in LAP. No visible inflammation was demonstrated for FLB, and the LAP system significantly increased the retention of FLB in the ocular tissues (compared to non-aggregated liposomes). Taking into account the capability of the novel LAP system to decrease inflammatory reactions towards calcein and FD4, and prolong the retention of FLB in ocular tissues, it is concluded that such systems, after further optimization, may be considered as promising effective and safe approaches for treatment of posterior segment ocular pathologies.

Prolonged retention of liposomes in the pleural cavity of normal mice and high tumor distribution in mice with malignant pleural effusion, after intrapleural injection.

Background: Intrapleural administration of compounds is a lung targeted, innovative therapeutic strategy for mesothelioma, which can be refined as a route for drug delivery that minimizes the potential for systemic toxicity. However, little is currently known about the retention of liposomal drugs at the site, after such topical administration. Purpose: To evaluate the retention of liposomes in lungs following intrapleural injection, and how this might be modulated by liposome properties and disease progression. Methods: DiR-incorporating liposomes with various lipid compositions and sizes were prepared, characterized (for size distribution and zeta potential) and injected intrapleurally in normal mice and mice with malignant pleural effusion (MPE). DiR retention in pleural cavity was followed by biofluorescence imaging. Results: Experimental results demonstrate that liposome size and PEG-coating, have a significant effect on residence time in the pleural cavity; negative surface charge does not. More than 20% liposomal-DiR is retained 24 d post-injection (in some cases), indicating the high potential towards localized diseases. Ex-vivo liposomal-DiR signal in tumors of MPE mice was similar to signal in liver, suggesting high tumor targeting potential of intrapleurally injected liposomes. Finally, no difference was noticed in liposomal-DiR retention between tumor-inoculated (MPE) and healthy mice, indicating the stability of liposomes in the presence of effusion (in MPE mice). Conclusion: The current study provides novel insights for using liposomes by intrapleural administration for the treatment of lung diseases.

Protection of dopamine towards autoxidation reaction by encapsulation into non-coated- or chitosan- or thiolated chitosan-coated-liposomes.

The aim of this work is to evaluate the potential of non-coated-, chitosan-(CS)- or chitosan-glutathione conjugate- (CS-GSH)-coated liposomes to protect the neurotransmitter Dopamine (DA) from the autoxidation reaction in neutral/alkaline conditions. This may be of interest in the development of nanotechnology-based approaches to improve Parkinson's disease treatment because decreased ROS production and reduced DA associated neurotoxicity are expected. For the mentioned purposes, DA-loaded vesicles were prepared by the Dried Reconstituted Vesicles (DRV) method, and were subsequently coated using solutions of polycations. As for the mean diameters of liposomes so prepared, the CS-GSH coated liposomes showed a significant decrease in size compared to the corresponding non-coated and CS-coated vesicles. The surface charge of DA-loaded non-coated liposomes was -10.8 mV, whereas the CS or CS-GSH coated vesicles showed a slightly positive ζ-potential. The capability of the herein studied vesicles to prevent DA autoxidation was evaluated by visual inspection, monitoring DA/lipid ratio as such and under stressed conditions. The results suggest that liposome formulations partially protect the neurotransmitter from the autoxidation reaction. In particular, the CS-GSH coated liposomes were more stable than the corresponding CS-coated and non-coated ones against the oxidative damage and were found to deliver the neurotransmitter in a sustained manner. Probably, this is due to the localization of the neurotransmitter in the core of the vesicles as indicated by XPS which confirmed the absence of the neurotransmitter on the surface of these vesicles.

Trypanocidal activity of arsonoliposomes: effect of vesicle lipid composition.

Sonicated arsonoliposomes were prepared using an arsonolipid with palmitic acid acyl chain (C16), mixed with phosphatidylcholine (PC-based) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC-based), and cholesterol (Chol) with a molar ratio C16 /PC or DSPC/ Chol 8:12:10. PEG-lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated to polyethylenoglycol 2000) containing vesicles (pegylated-arsonoliposomes) were also prepared. The in vitro and in vivo trypanocidal activity of the various types of arsonoliposomes was evaluated. Although PC-based arsonoliposomes exhibited in vivo activity on an acute trypanosomiasis animal model, no evidence of activity was demonstrated for DSPC-based or pegylated-arsonoliposomes on a chronic model. Despite the fact that DSPC-based and pegylated-arsonoliposomes have better bioavailability compared to PC-based ones, their in vitro activity is lower than that of PC-based arsonoliposomes, indicating the importance of arsonoliposome lipid composition on their trypanocidal activity and suggesting that further arsonoliposome structure design is required to overcome these disadvantages.

Sustained release of intravitreal flurbiprofen from a novel drug-in-liposome-in-hydrogel formulation.

A novel Flurbiprofen (FLB)-in-liposome-in-hydrogel formulation was developed, as a method to sustain the release and increase the ocular bioavailability of FLB following intravitreal injection. For this, FLB loading into liposomes was optimized and liposomes were entrapped in thermosensitive hydrogels consisted of Pluronic F-127 (P). FLB solution, liposomes, and FLB dissolved in hydrogel were also used as control formulations. Actively loaded liposomes were found to be optimal for high FLB loading and small size, while in vitro studies revealed that P concentration of 18% (w/v) was best to retain the integrity of the hydrogel-dispersed liposome, compared to a 20% concentration. The in vitro release of FLB was significantly sustained when FLB-liposomes were dispersed in the hydrogel compared to hydrogel dissolved FLB, as well as the other control formulations. In vivo studies were carried out in pigmented rabbits which were injected through a 27G needle with 1mg/mL FLB in the different formulation-types. Ophthalmic examinations after intravitreal injection of all FLB formulations, revealed no evidence of inflammation, hemorrhage, uveitis or endophthalmitis. Pharmacokinetic analysis results confirm that the hybrid drug delivery system increases the bioavailability (by 1.9 times compared to solution), and extends the presence of the drug in the vitreous cavity, while liposome and hydrogel formulations demonstrate intermediate performance. Furthermore the hybrid system increases MRT of FLB in aqueous humor and retina/choroid tissues, compared to all the control formulations. Currently the potential therapeutic advances of FLB sustained release formulations for IVT administration are being evaluated.

Physical stability of sonicated arsonoliposomes: effect of calcium ions.

The physical stability of sonicated arsonoliposomes in the absence and presence of Ca(2+) ions is evaluated. Cholesterol-containing arsonoliposomes composed of arsonolipids [having different acyl chains (C(12)-C(18))], or mixtures of arsonolipids with phospholipids (phosphatidylcholine or distearoyl-phosphatidylcholine) were prepared, and physical stability was evaluated in the absence and presence of CaCl(2), by vesicle dispersions turbidity measurements and cryo-electron microscopy morphological assessment. In some cases, vesicle zeta-potential was measured, under identical conditions. Results demonstrate that self-aggregation of the vesicles studied is low and influenced by the acyl chain length of the arsonolipid used, whereas calcium-induced aggregation is higher, correlating well with the decreased values of vesicle zeta-potential in the presence of Ca(2+) ions (weaker electrostatic repulsion). Acyl chain length of arsonolipids used has a significant quantitative effect on Ca(2+)-induced vesicle aggregation mainly for arsonoliposomes that contain phospholipids (mixed), compared with the vesicles that consist of plain arsonolipids (significant effect only for initial aggregation at time 0). Another difference between plain and mixed arsonoliposomes is that for mixed arsonoliposomes Ca(2+)-induced increases in turbidity are irreversible by ethylenediaminotetraacetic acid, suggesting that vesicle fusion is taking place. This was confirmed by cryo-electron microscopy observations. Finally, when phosphatidylcholine is replaced by distearoyl-phosphatidylcholine, arsonoliposomes are more stable in terms of self-aggregation, but in the presence of calcium, the turbidity and morphology results are similar.

The effect of pH on the electrophoretic behaviour of a new class of liposomes: arsonoliposomes.

Herein we report the effect of pH on the surface charge of a new class of liposomes: arsonoliposomes. Plain or mixed arsonoliposomes with cholesterol (Chol) and distearoyl-phosphatidylcholine (DSPC) in 1:1 molar ratio were prepared with lauryl-(C12), myristoyl-(C14) and palmitoyl-(C16) acyl side chain arsonolipids. The one step hydration method was used for vesicle preparation and zeta potential measurements were performed in the pH range from 3 to 9. The results revealed that these lipids hold a negative surface charge at all pH values investigated. The presence of cholesterol in 1:1 molar ratio results in higher zeta potential compared with plain arsonoliposomes with the exception of palmitoyl-(C16) acyl chain arsonolipids in neutral and slightly basic pH values. Oppositely, the DSPC (1:1 molar ratio) containing arsonoliposomes had lower values of zeta potential compared with plain arsonoliposomes. Concluding, the experimental results reveal that zeta potential of arsonoliposomes is indeed modified when the vesicles are incubated in environments with different acidity. In most cases these changes are in accordance with the ionization pattern of the arsonolipid headgroup, while some peculiar deviations may be connected with the known difference in the structure between some of the vesicle types studied.

Liposomes as vaccine carriers. Incorporation of soluble and particulate antigens in giant vesicles.

Giant liposomes (mean diameter 5.5 microns) composed of egg phosphatidylcholine or distearoyl phosphatidylcholine, phosphatidyl glycerol, cholesterol and triolein were prepared by a double emulsion technique. They were then mixed with model particulate (killed Bacillus subtilis, and killed Bacille Calmette-Guérin) and soluble (tetanus toxoid) vaccines and freeze-dried. Rehydration of the powder resulted in the generation of vesicles of similar mean diameter and diameter range, containing up to 27% (mean value) of the materials used for entrapment. Separation of entrapped from non-entrapped material was carried out by sucrose gradient centrifugation (B. subtilis and BCG) or centrifugation at 600 x g (toxoid). Light microscopy of liposomes containing B. subtilis labelled with fluorescein isothiocyanate revealed the presence of bacteria in individual vesicles which, in separate studies, were also found to entrap latex particles (0.5 and 1.0 micron diameter). Bacteria-containing liposomes could be freeze-dried in the presence of trehalose with most (83-87%) of the entrapped material recovered within the vesicles on reconstitution with saline. Liposomes were also shown to retain quantitatively their content of B. subtilis and, to a lesser extent, toxoid in the presence of mouse plasma at 37 degrees C and in situ after intramuscular injection into mice, for up to 24 h. Since liposomes are known (Gregoriadis, G. (1990) Immunol. Today 11, 89) to act as immunological adjuvants and vaccine carriers, giant vesicles containing microbes (live or attenuated if needed since the conditions of entrapment are mild) and, when appropriate, soluble antigens, could be used as multiple vaccines to ensure simultaneous presentation of antigens to immunocompetent cells.

Capability of arsonolipids to transport divalent cations: a Pressman cell study.

The ability of the newly synthesized arsonolipids (2,3-diacyloxyprophlarsonic acids) to transport cations was studied using the Pressman cell. Experimental results demonstrate that arsonolipids are much more efficient carriers of Ca(2+) and Mg(2+) than natural phosphatidic acid in the Pressman cell experiments. The ability of arsonolipids to transfer Ca(2+) is affected by the lipid side chain length in the order: C(12)>>C(14) approximately C(16). Ca(2+) is transferred faster than Mg(2+), suggesting that the latter is more tightly bound to the arsonolipids. The transfer kinetic curves are parabolic for C(12), while initially linear with a tendency to reach a steady state for C(14) and C(16), when the pH in the donor compartment was 8.3. The transport kinetics for both ions studied were best fitted by an equation derived from saturation kinetics that apply in reversible chemical reactions. The ion transfer rates increased as the pH in the donor compartment decreased.

Preparation and properties of arsonolipid containing liposomes.

Arsonolipids are analogs of phosphonolipids which have a chemically versatile head group. In preliminary cell culture studies, liposomes composed solely of arsonolipids or of phosholipid-arsonolipid mixtures, demonstrate a specific toxicity against cancer cells (Gortzi et al., unpublished results). The possibility of using such formulations as an alternative of arsenic trioxide with or without combination of other cytostatic agents (encapsulated in their aqueous interior) prompted the investigation of their physicochemical characteristics. Herein we compared the characteristics of arsonolipid containing vesicles with different lipid compositions. Experimental results and morphological observations reveal that non-sonicated formulations have different structures and stability (when both membrane integrity and aggregation are taken into account) depending on the acyl chain length of the arsonolipid. When phospholipids and especially cholesterol are included in their membranes almost all arsonolipids studied produce more stable vesicles. An interesting aspect of these arsonolipid containing vesicles is also their negative surface charge, which may be modulated by mixing phospholipids with arsonolipids. Sonicated vesicles have smaller sizes and profoundly higher stability, especially when containing cholesterol and phosphatidylcholine mixed with arsonolipids. The only exception is that of the arsonolipid with the C(12) acyl chain which was observed to produce long tubes which break down to cubes by sonication. In conclusion, these initial studies demonstrate that sonicated vesicles composed of arsonolipid and phospholipid mixtures mixed with cholesterol posses the stability required to be used as an arsonolipid delivery system. In addition, although cryo-electron microscopy demonstrated that the sonicated vesicles are elliptical in shape, their encapsulation efficiency is not significantly lower than sonicated phospholipid liposomes. Thereby, these vesicles may be also used for the delivery of other drug molecules which can be sufficiently retained in their aqueous interior.

Interactions of PC/Chol and PS/Chol liposomes with human cells in vitro.

Interactions between phosphatidylcholine (PC) or phosphatidylserine (PS) liposomes and human umbilical vein endothelial cells (HUVEC) or human promyelocytic leukemia cells (HL60) were investigated. Pyramine encapsulating or rhodamine incorporating small unilamellar liposomes with mean diameters around 80 nm (demonstrated to retain encapsulated material and to be nontoxic under experimental conditions) were used. Liposome uptake by both types of cells increased when increasing amounts of vesicles were co-incubated. For both lipid compositions, the interaction with HUVEC was very fast (association reached a plateau within 5 min) and so was the release of internalized vesicles (90% within 10 min at 37 degrees C). The reduced association values at 4 degrees C and the punctuate fluorescence observed in the cell cytoplasm after interaction, were indicative of whole liposome internalization. This internalization was clathrin-independent, since it was not inhibited by sodium azide and deoxyglucose. Pre-treatment of HUVEC with filipin or NEM resulted in modification of the interaction, something that could be due to alterations in the biochemical characteristics of HUVEC membranes that inhibit vesicular processes. In HL-60 cells, a slower association and faster release of PC/Chol liposomes was demonstrated, while association of both liposomes with these cells was energy-and temperature-independent. Nevertheless, morphological studies revealed differences in the interactions: A bright fluorescent rim observed after interaction with PC/Chol liposomes, suggests that these liposomes were adsorbed on the surface of HL60 cells, while the uniform cytoplasmic fluorescence observed after incubation with PS/Chol liposomes was indicative of fusion as the interaction mechanism.

Physicochemical properties of liposomes incorporating hydrochlorothiazide and chlorothiazide.

In an attempt to study the effect of hydrophobic drugs on liposome properties, multilamellar liposomes (MLV) consisting of phosphatidylcholine (PC) and incorporating chlorothiazide (CT) or hydrochlorothiazide (HCT), were prepared and characterized. Liposome size, surface charge, stability (in buffer, plasma and sodium cholate) and calcium-induced aggregation were studied for drug-incorporating liposomes and empty liposomes for comparison. Results show that drug incorporation affects liposome size, z-potential and stability in presence of buffer and plasma proteins. Indeed, drug-incorporating liposomes are slightly larger and have a negative surface charge, which increases with the amount of drug incorporated in the lipid membrane. The membrane integrity of drug incorporating liposomes (in absence and presence of plasma proteins) is significantly higher when compared with that of empty liposomes (for both drugs studied). On the contrary, vesicle membrane integrity in presence of sodium cholate and calcium induced vesicle aggregation, are not affected by drug incorporation. Leakage of thiazides from liposomes was demonstrated to be induced by dilution. Low amounts of thiazides (around 10-15%) are released when lipid concentration is over 0.1 mM, while further dilution increased drug leakage exponentially. Concluding, results demonstrate that the presence of HCT or CT in liposome membranes has a significant effect on main vesicle properties, which are known to influence vesicle targeting ability. Thereby, it is very interesting to continue studies in this respect, especially with more lipophilic drugs.

Characterization, stability and in-vivo distribution of asialofetuin glycopeptide incorporating DSPC/CHOL liposomes prepared by mild cholate incubation.

In this study, a small triantennary asialoglycopeptide of fetuin (A-F2) was used as a ligand to direct liposomes to hepatocytes. A-F2 was cleaved from asialofetuin, purified, conjugated with fatty acids and incorporated into pre-formed sonicated DSPC/Chol (2:1) liposomes. A mild cholate incubation method for incorporating the A-F2 ligand on pre-formed vesicles was used. In preliminary in vivo experiments 111In3+ encapsulated in A-F2/palmityl liposomes was seen to accumulate in the liver of mice significantly faster than when encapsulated in non-ligand bearing liposomes of the same lipid composition (studied before), justifying further investigation of this system. The presence of the A-F2/fatty acid conjugate in a functional form on the vesicle surface was confirmed by their reversible agglutination in the presence of Ricinus communis agglutinin (RCA120). Effects of ligand incorporation on the vesicle size distribution, z-potential, membrane integrity and stability were monitored. The results demonstrate that highest ligand incorporation was achieved when liposomes and ligand were co-incubated in the presence of 1 mM sodium cholate. Incorporation increased with the length of the fatty acid used for A-F2 conjugation. Ligand-bearing liposomes were demonstrated to be smaller in diameter (about 30%) with a more positive z-potential in comparison to control vesicles while ligand incorporation did not influence the liposome membrane integrity. The size of the ligand-incorporating vesicles was maintained after 24 hours of incubation in isotonic buffer, proving that the vesicles do not aggregate. Although the preliminary biodistribution results may suggest that ligand bearing liposomes are accumulating in the liver, further cell culture, in vivo distribution and especially liver fractionation studies are required in order to clarify the intrahepatic localization of these liposomes and the ability to target liver hepatocytes in vivo.

Effect of temperature and fat content on the solubility of hydrochlorothiazide and chlorothiazide in milk.

The solubility of hydrochlorothiazide and chlorothiazide in milk has been studied. Experiments were carried out at 5, 15, 25, and 37 degrees C on a buffer solution of pH 6.5, a 2.6% solution of casein, bovine skim milk samples, and bovine milk samples with fat contents of 0.75, 1.70, and 3.50%. The "total" solubility of both drugs in the media studied was higher than the buffer solubility. The highest "total" solubility for both drugs was observed in skim milk. Based on binding data of thiazides to milk, the "total" solubility was split into "free" and "bound" solubility. The increases of solubility noted cannot be explained on the basis of drug-milk binding data. The enhancement of solubility was attributed to the increase of intrinsic solubility of drugs in milk. Results of the thermodynamic analysis of solubility data showed that a different solubilization process of hydrochlorothiazide may be responsible for the high solubility values found in skim milk for this drug. In contrast, the thermodynamic parameters of chlorothiazide in all types of milk are similar, indicating a common solubilization mechanism. The biopharmaceutical significance of the findings is discussed in light of the freeze-dried drug-milk formulations and coadministration of drugs with milk in general.

Effect of temperature and fat content on the binding of hydrochlorothiazide and chlorothiazide to milk.

The binding of hydrochlorothiazide and chlorothiazide to milk has been measured. Experiments were carried out at 5, 15, 25, and 37 degrees C on bovine milk samples with fat contents of 0.75, 1.70, and 3.50%, using a wide range of drug concentrations to mimic concentrations encountered when a drug-milk freeze-dried system is utilized. Binding experiments with a 2.6% solution of casein were also carried out at the same temperature and concentration range of drugs. The binding to milk and casein was found to be not dependent on the concentration of drugs. The fat content of milk had no significant effect on the binding of both drugs. Higher binding was observed at lower temperatures than at higher temperatures for both drugs examined. The binding of both drugs to casein at 37 degrees C agrees fairly well with the corresponding binding to all types of milk at 37 degrees C. The potential significance of the findings in respect to preparation and in vivo delivery of drugs from drug-milk formulations is discussed.

Liposome-coated metal stents: an in vitro evaluation of controlled-release modality in the ureter.

PURPOSE: In vitro preparation of liposome-covered metal stents and loading of liposomal drug formulations that will slowly release the drug in the vicinity of the stent. MATERIALS AND METHODS: Polytetrafluoroethylene-coated stents were used. Large multilamellar (MLV) liposomes (phosphatidylcholine:cholesterol 1:1 mol/mol), empty or entrapping the corticosteroid anti-inflammatory drug, dexamethasone, were prepared by the thin-film hydration method and applied to pieces of stent using a simple and mild evaporation technique. Initially, a freeze-drying method for applying liposomes to stents was also evaluated, but it failed to produce stents that efficiently retain liposomal lipid when incubated in an aqueous environment. The presence of liposomes on the stent surface was confirmed by scanning electron microscopy. RESULTS: After analyzing the release of liposomal lipid (using a phospholipid assay) and liposomal drug (by a modified dexamethasone high-pressure liquid chromatography method) in an in vitro system developed to simulate in vivo conditions, it was found that 39.11+/-6.8% of the lipid and 50.84+/-5.48% of the drug was released from the stent pieces during 48 hours of incubation in the presence of artificial urine. The amount of dexamethasone released from stents during their application procedure was found to be negligible in an in vitro dry run. CONCLUSION: The use of stent-associated liposomal drug formulations as slow-release depots could be an efficient method of treating the untoward event of ureteral stent obstruction.