A Cationic Lipid Emulsion/DNA Complex as a Physically Stable and Serum-Resistant Gene Delivery System

Purpose. To develop a non-viral gene delivery system in the form ofan oil-in-water (o/w) lipid emulsionMethod. Cationic lipid emulsions were formulated with soybean oil,1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) as acationic emulsifier and other co-emulsifiers. The physicalcharacteristics of the lipid emulsion and the emulsion/DNA complex weredetermined. The in vitro transfection efficiency of the emulsion/DNAcomplex was determined in the presence of up to 90% serum. Results. The average droplet size and zeta potential of emulsions wereca. 180 nm and ca. +50 mV, respectively. Among the emulsions, astable formulation was selected to form a complex with a plasmidDNA encoding chloramphenicol acetyltransferase. By increasing theratio of emulsion to DNA, zeta-potential of the emulsion/DNA complexincreased monotonously from negative to positive without any changesin the complex size. The complex was stable against DNase I digestionand an anionic poly-l-aspartic acid (PLAA). The complex deliveredDNA into the cells successfully, and the transfection efficiency wasnot affected by complex formation time from 20 min to 2 h. Moreimportantly, the cationic lipid emulsion facilitated the transfer of DNAin the presence of up to 90% serum. Conclusions. The cationic lipid emulsion/DNA complex has physicalstability and serum resistant properties for gene transfer.     

Formulation Development and Antitumor Activity of a Filter-Sterilizable Emulsion of Paclitaxel

Purpose. Paclitaxel is currently administered i.v. as a slow infusion of asolution of the drug in an ethanol:surfactant:saline admixture. However,poor solubilization and toxicity are associated with this drug therapy.Alternative drug delivery systems, including parenteral emulsions, areunder development in recent years to reduce drug toxicity, improveefficacy and eliminate premedication. Methods. Paclitaxel emulsions were prepared by high-shearhomogenization. The particle size of the emulsions was measured by dynamiclight scattering. Drug concentration was quantified by HPLC and invitro drug release was monitored by membrane dialysis. The physicalstability of emulsions was monitored by particle size changes in boththe mean droplet diameter and 99% cumulative distribution. Paclitaxelpotency and changes in the concentration of known degradants wereused as chemical stability indicators. Single dose acute toxicity studieswere conducted in healthy mice and efficacy studies in B16 melanomatumor-bearing mice. Results. QW8184, a physically and chemically stable sub-micronoil-in-water (o/w) emulsion of paclitaxel, can be prepared at high drugloading (8-10 mg/mL) having a mean droplet diameter of <100 nmand 99% cumulative particle size distribution of <200 nm. In vitro release studies demonstrated low and sustained drug release both inthe presence and absence of human serum albumin. Based on singledose acute toxicity studies, QW8184 is well tolerated both in miceand rats with about a 3-fold increase in the maximum-tolerated-dose(MTD) over the current marketed drug formulation. Using the B16mouse melanoma model, a significant improvement in drug efficacywas observed with QW8184 over Taxol^®. Conclusions. QW8184, a stable sub-micron o/w emulsion of paclitaxelhas been developed that can be filter-sterilized and administered i.v.as a bolus dose. When compared to Taxol^®, this emulsion exhibitedreduced toxicity and improved efficacy most likely due to thecomposition and dependent physicochemical characteristics of the emulsion.     

Effect of Non-Ionic Surfactants on the Formation of DNA/Emulsion Complexes and Emulsion-Mediated Gene Transfer

Purpose. To study the structure-function relationship of non-ionic surfactants in emulsion-mediated gene delivery. Methods. Four different types of non-ionic surfactants including Tween, Span, Brij and pluronic copolymers were used as co-emulsifiers for preparation of emulsions composed of Castor oil, dioleoylphosphatidylethanolamine (DOPE) and 3[N-(N, N-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol). The effect of different surfactants on the formation of DNA/emulsion complexes and transfection activity were analyzed using plasmid DNA containing luciferase cDNA as a reporter gene. Results. Non-ionic surfactants containing branched polyoxyethylene chains as the hydrophilic head group were more effective in preventing the formation of large DNA/emulsion complexes than those containing one or no polyoxyethylene chain. All emulsion formulations except those containing Brij 700 exhibited high activity in transfecting mouse BL-6 cells in the absence of serum. In the presence of serum, however, transfection activity of each formulation varied significantly. Emulsions containing Tween, Brij 72, pluronic F68 and F127 demonstrated increased activity in transfecting cells in the presence of 20% serum. In contrast to emulsions containing Span, long chain polyoxyethylene of Brij showed decreased transfection activity. The particle size of the DNA/emulsion complexes and their ability to transfect cells are dependent on the concentration of non-ionic surfactant in the formulation. Conclusions. The structure of the hydrophilic head group of the non-ionic surfactants in the emulsion is important in determining how DNA molecules interact with emulsions and the extent to which DNA is transferred inside the cell.     

Long-Circulating Emulsions (Oil-in-Water) as Carriers for Lipophilic Drugs

Purpose. Rapid clearance of parenterally administered oil-in-water emulsions from blood by the reticuloendothelial system (RES), mainly macrophages of the liver and spleen, has been one of the major obstacles for delivering lipophilic drugs to cells other than those in the RES. The purpose of this study therefore is to overcome this problem and develop emulsions that will have prolonged blood circulation time. Methods. A series of amphipathic polyethylene-glycol (PEG) derivatives have been included as co-emulsifier into emulsions composed of Castor oil and phosphatidylcholine. The effect of amphipathic PEG on reducing the RES uptake and prolonging the blood circulation of the emulsion particles has been tested in vivo using mice as an animal model. Results. Inclusion of PEG derivatives such as Tween-80 or dioleoyl N-(monomethoxy-polyethyleneglycol succinyl)phosphotidylethanolamine (PEG-PE) into emulsions composed of Castor oil and phosphatidylcholine decreases the RES uptake and increases blood residence time of the emulsions. The activity of PEG derivatives in prolonging the circulation time of emulsions depends on the PEG chain length (PEG2000PEG5000>PEG1000, Tween-80) and the PEG density on emulsion surface. Conclusions. Inclusion of amphipathic PEG as emulsifier into oil-in-water emulsions is a very effective method to prolong the blood half life of the emulsions. Emulsions with long circulating half life in blood should be very useful as a delivery vehicle for lipophilic drugs.     

Interfacial Properties as Stability Predictors of Lecithin-Stabilized Perfluorocarbon Emulsions

ABSTRACT

The purpose of this study was to determine whether the addition of small quantities of minor lecithin components (phosphatidylinositol, phosphatidic acid, lysophosphatidylethanolamine, and cholesterol) and Pluronic F68 to lecithin could improve the stability of lecithin-stabilized perfluorocarbon emulsions. Attempts were made to correlate emulsion stability with interfacial properties (tension and charge). Dynamic interfacial tension was determined using a Teflon Wilhelmy plate method [reported previously (1)]. Emulsions were prepared by microfluidization. Microelectrophoresis was used to measure emulsion droplet charge, and photon correlation spectroscopy and Coulter analysis were used to determine emulsion stability as a function of droplet size. Thermal kinetic accelerated stability testing was conducted. Various droplet size parameters were used to compare emulsion stabilities, and an overall stability ranking, based on these parameters, was obtained for each emulsion. Small quantities of additives altered emulsion stability and these data were correlated with interfacial properties and initial droplet diameters. The addition of cholesterol to lecithin resulted in the most stable perfluorocarbon emulsion.     

Enhanced Tumor Delivery and Antitumor Activity of Palmitoyl Rhizoxin Using Stable Lipid Emulsions in Mice

Purpose. A highly lipophilic antitumor agent, 13-O-palmitoyl-rhizoxin (RS-1541), was incorporated into lipid emulsions of various sizes consisting of triglyceride ODO and surfactant HCO-60. Pharmacokinetics, toxicities, and antitumor activities were evaluated after intravenous administration to mice bearing subcutaneously inoculated M5076 sarcoma cells. Methods. The levels of RS-1541 in the plasma and tissues including tumor, were determined by HPLC. The maximum tolerated dose (MTD) was estimated by toxic death and change in body weight. The decrease in tumor diameter was measured for antitumor activity. Results. There existed large variations in pharmacokinetics of RS-1541, depending on the size of emulsion particles. Compared with a colloidal solution (reference solution), the small (110nm) and medium (230nm) size emulsions showed high concentrations of RS-1541 in the tumor, while the large emulsions (350nm–630nm) exhibited low concentrations. The MTD of RS-1541 was reduced, when incorporated in the emulsions larger than 220nm in size. At MTD, each size of emulsions (70nm–380nm) effectively retarded the tumor growth and increased survival time. The maximum effect was achieved for the 220 nm emulsions. Conclusions. When particle size is properly selected, these emulsions could be promising and effective as an injectable carrier for lipophilic antitumor agents in order to enhance the tumor delivery and efficacies while reducing toxicities.     

A Novel Phase Inversion-Based Process for the Preparation of Lipid Nanocarriers

Purpose. To develop and subsequently evaluate a novel phase inversion-based method used to formulate lipidic nanocapsules. Methods. Mechanical properties of emulsions prepared by multi-inversion phase processes were investigated using a drop tensiometer. Based on the results obtained, a formulation process was developed and a new type of nanocarrier was prepared. These particulates were sized by photon correlation spectroscopy and were visualized by atomic force microscopy and transmission electronic microscopy. Differential scanning calorimetry was also performed. Results. The marginally cohesive but stable interfacial properties of the initial system led to the formulation of lipidic nanocapsules that were composed of a liquid core surrounded by a cohesive interface and were dispersed in an aqueous medium. These related suspensions were stable upon dilution for several months. The control of the formulation parameters allowed an adjustment of the particle mean diameter in the range of 25-100 nm with a monodisperse size distribution. Conclusions. A novel and convenient process for the preparation of lipidic nanocapsules is described. The structure of these particulates resembles a hybrid between polymeric nanocapsules and liposomes. Such nanocapsules display a strong potential for drug delivery.     

Studies on the effect of pilocarpine incorporation into a submicron emulsion on the stability of the drug and the vehicle.

In order to obtain a novel ocular formulation with a potential for prolonging pilocarpine activity, the drug (2.0%) was incorporated into a submicron emulsion containing soya-bean oil and lecithin as emulgator. The effect of drug incorporation into the emulsion on its physical stability and on the other hand, the potential of the vehicle to reduce drug degradation at pH higher than 5.0 was studied. The pH was adjusted to 6.5 or 5.0 and the physicochemical stability of the formulations was observed. The mean diameter of oily particles in the resulting emulsions measured by a laser diffractometer was 0.6-0.7 micron and this was larger than in a drug-free emulsion where a 0.33 micron value was measured. The formulations were physically stable for 6 months at 4 degrees C, but progressing chemical degradation of pilocarpine was noted at pH 6.5. At that pH nearly 8% of pilocarpine was degraded to isopilocarpine and pilocarpic acid, both in the emulsion and in the solution. Thus, it may be concluded that pilocarpine in submicron emulsion is not protected against degradation. The presence of pilocarpine changes the physical stability of the vehicle since the formulation was easily destabilized during autoclaving or at room temperature. In the presence of higher concentration of lecithin (2.4%) or co-emulgators (poloxamer 2.0% or Tween 80 0.5%) the mean droplet size in the emulsions was the same as in a drug-free system. However the emulsions containing poloxamer were not stable during storage. Viscosity of pilocarpine emulsions can be increased by addition of methylcellulose or sodium carmellose (1.0%), but an intensive creaming occurs in these systems. Pilocarpine base is less suitable for emulsion preparation than hydrochloride salt, and emulsions prepared at pH 5.0 show the most satisfying stability.     

Effect of methylcellulose on the stability of oil-in-water emulsions

The objective of this study was to investigate how polymers used as auxiliary emulsifiers improve the stability of oil-in-water emulsions. One stable emulsion and three unstable emulsions were formulated with 30% mineral oil and an emulsifier blend of Tween® 40 and Span® 20. The stable emulsion (SE) contained 2% emulsifier blend optimized for maximum stability. One unstable emulsion, UEI, was formulated to contain 0.5% of the same emulsifier blend as the SE formulation. Two unstable emulsions were formulated to contain an unbalanced emulsifier blend, one with excessive hydrophilic emulsifier (UE2) and one with excessive lipophilic emulsifier (UE3). A series of emulsions was prepared containing increasing amounts of methylcellulose for each base emulsion. Creaming and change in particle size were measured to evaluate stability. The addition of the polymer to the stable emulsion caused instability leading to creaming and eventual oil separation. This effect of the polymer was more pronounced in UEI emulsions. However, the addition of the polymer improved the stability of the UE2 and UE3 series of emulsions. The polymer also caused a reduction in the particle size of UE3 emulsions and a proportionally larger increase in the viscosity of UE2 emulsions. These results suggest that (i) methylcellulose could act as a hydrophilic emulsifier only in the absence of Tween^® 40, (ii) methylcellulose and Tween^® 40 associate to form a complex and (iii) the concentration of Tween^® 40 is the determining factor for the stability of emulsions. A model of the methylcellulose-Tween^® 40 association and its effect at the mineral oil-water interface is proposed.     

Study of the Breakup Under Shear of a New Thermally Reversible Water-in-Oil-in-Water (W/O/W) Multiple Emulsion

Purpose. Thickening of the external aqueous phase of W/O/W multiple emulsions is essential to increase the release under shear. However, it leads to globules bursting during fabrication. To reduce this problem, we have tested a novel thermally reversible hydrogel, EMP hydrogel. This way, the corresponding multiple emulsion (EMPME) would gel only at skin temperature, which may increase the active ingredient delivery when topically applied. Methods. Samples were sheared at different shear rates and temperatures (20, 30, and 35°C) with a controlled rheometer. A granulometric analysis was then performed with a laser diffraction granulometer, to assess the break up as a function of the shear rate at the three temperatures. Conductometric measurements (CDM 230 conductometer) provided the corresponding release curves. Results. As we expected, EMPME exhibited a thermally reversible behavior. Compared to a reference emulsion thickened by carbopol, this new thermo–sensitive multiple emulsion displayed higher break up and fraction released at 35°C. Conclusion. The first thermally reversible multiple emulsion has been developed in the present work. This one presents interesting advantages: (1) an easy fabrication process with a higher entrapment yield and (2) a higher fraction released at 35°C compared with the reference emulsion.     

Fat Emulsions Based on Structured Lipids (1,3-specific triglycerides): An Investigation of the in Vivo Fate

Purpose. Structured lipids (1,3-specific triglycerides) are new chemical entities made by enzymatic transesterification of the fatty acids in the 1,3 positions of the triglyceride. The purpose of this study is to characterize structured lipids with either short chain fatty acids or medium chain fatty acids in the 1,3 positions with regard to their hydrophobicity, and investigate the in vivo fate in order to evaluate the potential of structured lipids as core material in fat emulsions used as parenteral drug delivery system. Methods. The lipids were characterized by employing reversed phase high performance liquid chromatography. The biodistribution of radio-actively labeled emulsions was studied in rats. Results. By employing high performance liquid chromatography a rank order of the hydrophobicities of the lipids could be given, with the triglycerides containing long chain fatty acids being the most hydrophobic and the structured lipid with short chain fatty acids in the 1,3 positions the least. When formulated as fat emulsions, the emulsion based on structured lipids with short fatty acids in the 1,3 positions was removed slower from the general blood circulation compared to emulsions based on lipids with long chain fatty acids in the 1,3 positions. Conclusions. The type of core material influences the in vivo circulation time of fat emulsions.     

DRV Liposomal Bupivacaine: Preparation,Characterization, and In Vivo Evaluation in Mice

Purpose. To evaluate the dehydration-rehydration technique to prepare a formulation of liposomal bupivacaine, and to assess its analgesic efficacy. Methods. Bupivacaine hydrochloride (BUP) was encapsulated into dehydration-rehydration vesicles (DRV) of varying phospholipid (PL) compositions. Two bilayer-forming phospholipids were used, the fluid dimyristoyl-phosphatidylcholine and the solid dis- tearoyl-phosphatidylcholine (DSPC), with 20 or 40 mol% cholesterol, in the presence of bupivacaine at a 1.28 or 0.64 BUP/PL mole ratio. After rehydration, drug/lipid ratios were determined. The formulation with the highest drug/lipid ratio (DSPC/cholesterol in an 8:2 mole ratio prepared in the presence of bupivacaine in a 1.28 BUP/PL mole ratio) was adjusted to a final bupivacaine concentration of 3.5% or 0.5%. The duration of skin analgesia after subcutaneous injection in mice produced by these formulations was compared with the conventional administration of a plain 0.5% solution of BUP. In addition, the concentration of residual bupivacaine at the injection site was followed for 96 h. Results. The relatively low organic solvent/aqueous phase and membrane/aqueous phase partition coefficients, together with liposomal trapped volume and BUP/PL mole ratio, indicated that most of the drug was encapsulated in the intraliposome aqueous phase of the DRV. The DSPC/cholesterol 8:2 mole ratio had the best drug encapsulation (BUP/PL = 0.36). Compared to plain BUP, these BUP-DRV produced significant prolongation of analgesia, which is explained by longer residence time of the drug at the site of injection. Conclusions. Bupivacaine-DRV may have a role in achieving safe, effective, and prolonged analgesia in humans.     

Melatonin-based pickering emulsion for skin's photoprotection

Abstract

Context: Based on its antioxidant activity, melatonin was recently found to have a protection effect against photocarcinogenesis.

Objective: This work aimed to develop an innovative sunscreen formulation based on the Pickering emulsions concept, stabilized by physical UV filters, modified starch and natural oils associated to melatonin as a key strategy for prevention against UV-induced skin damage.

Materials and methods: For this purpose, melatonin was incorporated in Pickering emulsions that were characterized using physicochemical, in vitro and in vivo testing. Physicochemical studies included physical and chemical stability by a thorough pharmaceutical control. The possible protective effects of melatonin against UV-induced cell damage in HaCaT cell lines were investigated in vitro. The safety assessment and the in vivo biological properties of the final formulations, including Human Repeat Insult Patch Test and sunscreen water resistance tests were also evaluated.

Results and discussion: These studies demonstrated that melatonin sunscreen Pickering emulsion was beneficial and presented a powerful protection against UVB-induced damage in HaCat cells, including inhibition of apoptosis. The inclusion of zinc oxide, titanium dioxide, green coffee oil and starch ensured a high SPF (50+) against UVA and UVB.

Conclusion: The combination of melatonin, multifunctional solid particles and green coffee oil, contributed to achieve a stable, effective and innovative sunscreen with a meaningful synergistic protection against oxidative stress.     

Synthesis of a Lipophilic Daunoruhicin Derivative and Its Incorporation into Lipidic Carriers Developed for LDL Receptor-Mediated Tumor Therapy

Purpose. Many tumors express elevated levels of LDL receptors (apoB, E receptors) on their membranes. Selective delivery of anti-neoplastic drugs to tumors by incorporation of these drugs into LDL or LDL-resembling particles should improve the efficacy of tumor therapy and minimize the severe side-effects. Since the apolipoproteins on the particles are essential for the LDL receptor recognition, drugs should preferably be incorporated into the lipid moiety. Most anti-tumor agents are too hydrophilic for incorporation into these carriers. Methods. We synthesized LAD, a lipophilic prodrug of daunorubicin, by coupling the drug via a lysosomally degradable peptide spacer to a cholesteryl oleate analog. Results. The overall yield of the synthesis was 50% with a purity of >90%. Radioactively ([³H]) labeled LAD was obtained via a slightly modified procedure (yield 40%). The octanol/water partition coeffient of LAD is 30-fold higher than that of daunorubicin. LAD could be incorporated into triglyceride-rich lipid emulsions and small liposomes, which, if provided with apoE, have been demonstrated earlier to be cleared in vivo via the LDL receptor. The liposomes contained approximately 10 molecules of LAD per liposomal particle. Analysis of differently sized LAD-containing emulsions suggests that LAD associates with the surface of lipidic particles. In the presence of human serum, LAD did not dissociate from the emulsion particles, indicating a firm association of LAD with the carrier. Conclusions. The coupling of a cholesterol ester analog to daunorubicin results in a lipophilic prodrug that can be firmly anchored into lipidic carriers. LAD-loaded emulsions and liposomes provided with recombinant apoE will be tested in the near future for their ability to deliver LAD to tumor tissue in vivo via the LDL receptor.     

Stability of parenteral nanoemulsions loaded with paclitaxel: the influence of lipid phase composition,drug concentration and storage temperature

Abstract

Paclitaxel was loaded into licensed parenteral nutrition nanoemulsions (Clinoleic® and Intralipid®) using bath sonication, and the stability of the formulations was investigated following storage for two weeks at room temperature or at 4?°C. In general, Clinoleic droplets were smaller than Intralipid droplets, being around 255 and 285?nm, respectively, for blank and freshly loaded emulsions. Regardless of storage temperature, the Clinoleic exhibited a very slight or no increase in droplet size upon storage, whilst the droplet size of the Intralipid emulsion increased significantly. The droplet size of both emulsions was minimally affected by paclitaxel concentration within the range of 0, 1, 3 and 6?mg/ml. The pH of both emulsions markedly decreased upon storage at room temperature, which was possibly attributed to the production of fatty acids resulting from phospholipid hydrolysis. However, at 4?°C, the pH of Clinoleic emulsion was unaffected by storage or paclitaxel concentration while the Intralipid emulsion demonstrated a trend for pH reduction. Both nanoemulsions had a negative zeta potential, with the Clinoleic formulations having the highest charge, possibly explaining the better size stability of this emulsion. Overall, this study has shown that paclitaxel was successfully loaded into clinically licensed parenteral emulsions and that Clinoleic showed greater stability than the Intralipid.     

Effect of Nonionic Surfactant on Transport of Model Drugs in Emulsions

Purpose. To investigate the influence of excess surfactant on transport kinetics in emulsions, using phenylazoaniline (PAA), benzocaine, benzoic acid and phenol as model drugs. Mineral oil was chosen as the oil phase and the nonionic surfactant, polyoxyethylene oleyl ether (Brij 97) as the emulsifier. Methods. Model drug transport in emulsions was investigated using side by side diffusion cells mounted with hydrophilic dialysis or hydrophobic membranes. A novel method, involving a combination of a membrane equilibrium technique and surface tension measurement (Wilhelmy plate method), was developed to determine surfactant critical micelle concentration (CMC) in the presence of O/W emulsions. Emulsion stability was determined by droplet size analysis as a function of time, temperature and dilution using photon correlation spectroscopy and a light blockage technique. Model drug mineral oil/water partition coefficients and aqueous solubilities were determined in the presence of surfactant. Results. The emulsion CMC value was used to calculate micellar phase concentration. The transport rates of PAA and benzocaine in emulsions increased with increase in Brij 97 micellar concentration up to 1.0 % w/v and then decreased at higher surfactant concentrations. The transport rates of the more hydrophilic compounds, benzoic acid (ionized form, pH 7.0) and phenol, were not affected by the presence of micellar phase. Conclusions. Excess surfactant affected the transport rates of the model drugs in the emulsions depending on drug lipophilicity. Transport rates measured using side by side diffusion cells appeared to be governed by model drug partitioning rates from the oil to the continuous phases and by membrane type.     

Effect of Particle Size and Charge on the Disposition of Lipid Carriers After Intratumoral Injection into Tissue-isolated Tumors

Purpose. Pharmacokinetic properties of various lipid carriers (liposome and emulsions) after intratumoral injection were studied in perfusion experiments using tissue-isolated tumor preparations of Walker 256 carcinosarcoma. Methods. Four types of lipid carriers, large emulsion (254 nm), small emulsion (85 nm), neutral liposomes (120 nm) and cationic liposomes (125 nm) were prepared. We quantified their recovery from the tumor, leakage from the tumor surface and venous outflow after intratumoral injection into perfused tissue-isolated tumors, and analyzed venous appearance curves based on a pharmacokinetic model. Results. In contrast to the small emulsion and neutral liposomes, which immediately appeared in the venous outflow perfusate following intratumoral injection, the appearance of the cationic liposomes and the large emulsion was highly restricted, clearly demonstrating that intratumoral clearance of these formulations can be greatly retarded by the cationic charge and large particle size, respectively. The venous appearance rate-time profiles were fitted to equations derived from a two-compartment model by nonlinear regression analysis. When the calculated parameters were compared among these four formulations, the venous appearance rate did not exhibit such a large difference; however, the rate of transfer from the injected site to the compartment which involves clearance by venous outflow was all very different. Conclusions. The results of this study indicate that the determining factor which alters the pharmacokinetic properties of these lipid carriers after intratumoral injection is not the rate of transfer from the interstitial space to the vascular side but the rate of intratumoral transfer from the injection site to the well-vascularized region.     

A Novel Formulation of VIP in Sterically Stabilized Micelles Amplifies Vasodilation In Vivo

Purpose. To determine whether human vasoactive intestinal peptide (VlP)-poly(ethylene glycol) (PEG)-grafted distearoyl-phosphatidyleth-anolamine (DSPE) micelles elicit potent and stable vasodilation in vivo. Methods. PEG-DSPE micelles were prepared by co-precipitation. VIP was loaded into micelles by incubation at room temperature. Vasoactivity of VIP in SSM was determined by monitoring changes in diameter of resistance arterioles in the in situ hamster cheek pouch using intravital microscopy. Results. VIP easily undergoes self-assembly into small PEG-DSPE micelles (mean [±SEM] size, 18 ± 1 nm) in a time-dependent fashion. This generates a potent vasoactive matrix at nanomole concentrations of VIP as manifested by ~3-fold potentiation and prolongation of vasodilation relative to that evoked by aqueous VIP alone (p < 0.05). This response is specific and mediated by the L-arginine/nitric oxide (NO) biosynthetic pathway. Micellar VIP dispersion remains vasoactive for at least 14 days after preparation and storage at 4°C. Conclusions. A novel, self-associated, small and stable PEG-DSPE micellar formulation of VIP amplifies vasodilation in the in situ peripheral microcirculation in a specific fashion by elaborating NO. An optimized formulation could be considered for certain cardiovascular disorders associated with L-arginine/NO biosynthetic pathway dysfunction.     

pH-Metric logP 10. Determination of Liposomal Membrane-Water Partition Coefficients of lonizable Drugs

Purpose. To investigate a novel approach for the determination of liposomal membrane-water partition coefficients and lipophilicity profiles of ionizable drugs. Methods. The measurements were performed by using a pH-metric technique in a system consisting of dioleylphosphatidylcholine (DOPC) unilamellar vesicles in 0.15 M KC1 at 25°C. The DOPC unilamellar vesicle suspension was prepared via an extrusion process. Results. The liposomal membrane-water partition coefficients of eight ionizable drugs: ibuprofen, diclofenac, 5-phenylvaleric acid, warfarin, propranolol, lidocaine, tetracaine and procaine were determined and the values for neutral and ionized species were found to be in the ranges of approximately 4.5 to 2.4 and 2.6 to 0.8 logarithmic units, respectively. Conclusions. It has been shown that the liposomal membrane-water partition coefficients as derived from the pH-metric technique are consistent with those obtained from alternative methods such as ultrafiltration and dialysis. It was found that in liposome system, partitioning of the ionized species is significant and is influenced by electrostatic interaction with the membranes. We have demonstrated that the pH-metric technique is an efficient and accurate way to determine the liposomal membrane-water partition coefficients of ionizable substances.     

Inhibition of liver metastasis by all-trans retinoic acid incorporated into O/W emulsions in mice

All-trans retinoic acid (ATRA) was incorporated into lipid emulsions in an attempt to alter its distribution characteristics and improve its inhibition of liver cancer metastasis. Lipid emulsions composed of egg phosphatidylcholine, cholesterol, and soybean oil were the optimized carriers for ATRA delivery, as shown by the submicron particle size and high incorporation efficiency. The particle size and zeta potential of ATRA incorporated into emulsions were about 133 nm and -11 mV, respectively. In vitro drug release study demonstrated that the release of ATRA from emulsions was sustained in the absence and present of bovine serum albumin, suggesting that ATRA was stable when incorporated in emulsions. After intravenous administration in mice, [3H]cholesteryl hexadecyl ether incorporated into emulsion, which is the inherent distribution of emulsions, accumulated gradually mainly in the liver. The blood concentration and hepatic accumulation of [3H]ATRA incorporated into emulsion was significantly higher than that of serum dissolving [3H]ATRA, which represent the original distribution characteristic of free ATRA. In a murine liver metastasis model by colon adenocarcinoma, the liver metastasis number and liver weight were significantly reduced and the survival time of mice was prolonged following intravenous injection of ATRA incorporated into emulsions.