Reverse Phase Evaporation Technique Research Articles

Characterization and in-vivo ocular absorption of liposome-encapsulated acyclovir.

The potential of liposomes as an in-vivo ophthalmic drug delivery system for acyclovir was investigated. The drug-membrane interaction was evaluated by means of differential scanning calorimetry analysis. These experiments showed that acyclovir is able to interact with both positively and negatively charged membranes via electrostatic or hydrogen bonds. No interaction was observed with neutral membranes made up of dipalmitoylphosphatidylcholine. Different liposome preparation procedures were carried out to encapsulate acyclovir. The drug encapsulation mainly depends on the amount of water which the liposome system is able to entrap. In the case of multilamellar vesicles, charged systems showed the highest encapsulation efficiency. No particular difference in the encapsulation efficiency was observed for oligolamellar vesicles prepared with the reverse-phase evaporation technique. Oligolamellar liposomes showed the highest acyclovir encapsulation parameters and had release profiles similar to those of multilamellar liposomes. In-vivo experiments using male New Zealand albino rabbits were carried out to evaluate the aqueous humour concentration of acyclovir bioavailability. The most suitable ophthalmic drug delivery system was oligolamellar systems made up of dipalmitoylphosphatidylcholine-cholesterol-dimethyldioctadecyl glycerole bromide (7:4:1 molar ratio), which presented the highest encapsulation capacity and were able to deliver greater amounts of the drug into the aqueous humour than a saline acyclovir solution or a physical liposome/drug blend.

Preparation and lacrimal pharmacokinetics of eye drops of puerarinliposomes in rabbit tears

To prepare eye drops of puerarin liposomes and investigate its lacrimal pharmacokinetics in rabbit tears. Puerarin liposomes were prepared by reverse phase evaporation technique. The liposomes and free puerarin were separated by SephadexG-50 chromatography and then encapsulation ratio of liposomes was determined in detail. Micromorphology of liposome particles was observed by electronic transmission microscope and the size distribution of the liposomes was analyzed by laser particle size analyzer. The concentration of puerarin in rabbit's tears was determined by HPLC after ocular administration of 50 microL puerarin liposomes while puerarin eye drops was chosen as control with the same puerarin concentration. The pharmacokinetic parameters were calculated by software program 3P97 according to one-compartment mode. Global liposome nanoparticles with diameter of about 195.7 nm were prepared successfully. The encapsulation ratio of puerarin in the liposomes was 48.3%. The mean residence time (MRT) value and the area under concentration (AUC) of puerarin in liposome were 3.89 and 3.06 times more than those of puerarin eye drops. Liposomes as a drug carrier can greatly increase the concentration of puerarin in tears, enhance the pre-ocular retention time than that of eye drops.

Liposomes and Micellar Dispersions For Delivery of Benzoheterocyclic Derivatives of Distamycin A

In this article we describe the production and characterization of specialized delivery systems for some distamycin derivatives (DD), namely liposomes and micellar dispersions. All the formulations were designed to increase the solubility of DD in an aqueous environment and to reduce the possible toxicity problems related to the administration of these drugs. For instance, liposomes were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters, then characterized in terms of dimensions, morphology, and encapsulation efficacy. The analysis of their in vitro antiproliferative activity on cultured human and mouse leukemic cells demonstrated that liposomes and micellar dispersions containing DD exert quite different effects. These effects were compared with those shown by the free drug depending on type of drug and also cell line used.

Solubilisation and transformation of amphipathic lipidic dendron vesicles (dendrisomes) into mixed micelles

A novel lipidic peptide dendron was synthesised using solid-phase techniques. To a third-generation polylysine dendron were coupled eight α-amino tetradecanoic acid (C 14) molecules to the eight Nα and Nɛ of the terminal lysines and three pendant C 14 chains were coupled to the C-terminal of the starting lysine. This novel dendron was found to self-assemble in water into vesicular structures or “dendrisomes”. These dendrisomes were prepared using a reverse-phase evaporation (REV) technique and visualised by transmission electron microscopy (TEM). The interaction of these second order structures with the non-ionic surfactant iso-octyl phenyl poly(10)oxyethylene ether (Triton X-100) was studied by turbidimetry, photon correlation spectroscopy (PCS) and TEM. Quantitative estimation of the solubilised peptide dendron was carried out by the bicinchoninic acid (BCA) assay. The dendrisomes are transformed into surfactant–dendron mixed micelles. The dispersion became optically clear at a concentration 8 mM (13.4:1 Triton X-100:dendron molar ratio). When the concentration of the surfactant was increased up to 60–100 mM (100–170:1 Triton X-100:dendron molar ratio) the aggregate diameter decreased to the micellar range, namely 12–50 nm. At a 100:1 Triton X-100:dendron molar ratio, the BCA assay showed complete dendron solubilisation. This suggests that the cationic lipidic dendron and their assemblies can be solubilised by amphipathic non-ionic moieties when the surfactant–dendron interaction is more energetically favourable than dendron–dendron and surfactant–surfactant interactions.

Investigation of lectin-modified insulin liposomes as carriers for oral administration

The aim of this study was to design and characterize lectin-modified liposomes containing insulin and to evaluate the potential of these modified colloidal carriers for oral administration of peptide and protein drugs. Wheat germ agglutinin (WGA), tomato lectin (TL), or Ulex europaeus agglutinin 1 (UEA1) were conjugated by coupling their amino groups to carbodiimide-activated carboxylic groups of N-glutaryl-phosphatidylethanolamine ( N-glut-PE). Insulin liposomes dispersions were prepared by the reverse-phase evaporation technique and modified with the lectin- N-glut-PE conjugates. Lectin-modified liposomes were characterized according to particles size, zeta potential and entrapment efficiency. The hypoglycemic effect indicated by pharmacological bioavailability of insulin liposomes modified with WGA, TL and UEA1 were 21.40, 16.71 and 8.38% in diabetic mice as comparison with abdominal cavity injection of insulin, respectively. After oral administration of the insulin liposomes modified with WGA, TL and UEA1 to rats, the relative pharmacological bioavailabilities were 8.47, 7.29 and 4.85%, the relative bioavailability were 9.12, 7.89 and 5.37% in comparison with subcutaneous injection of insulin, respectively. In the two cases, no remarkable hypoglycemic effects were observed with the conventional insulin liposomes. These results confirmed that lectin-modified liposomes promote the oral absorption of insulin due to the specific-site combination on GI cell membrane.

Liposomal amikacin dry powder inhaler: Effect of fines on in vitro performance

The aim of the present investigation was to prepare and evaluate the influence of adding fines on the in vitro performance of liposomal amikacin dry powder inhaler (AMK LDPI) formulations. Liposomes composed of hydrogenated soyaphosphatidylcholine, cholesterol and saturated soyaphosphatidylglycerol (AMK 1), or stearylamine (AMK 2) were prepared by a reverse phase evaporation technique, extruded to reduce size and separated from unentrapped drug. Purified liposomal dispersion was subjected to lyophilization using optimized cryoprotectant to achieve maximum percentage drug retention (PDR). Lactose carrier in varying mass ratios with or without addition of fines in different mixing sequences was used to formulate AMK LDPI formulations. AMK LDPI formulations were characterized for angle of repose, compressibility index, dispersibility index, scanning electron microscopy, and fine particle fraction (FPF). PDR was found to be 97.6% +/- 2.2% for AMK1 and 98.5% +/- 1.9% for AMK2 using sucrose as optimized cryoprotectant in lipid:sucrose ratio of 1:4. Lactose carrier containing 10% fines (wt/wt) was found to be the optimum blend at 1:5 mass ratio of liposome:lactose. The addition of fines and the order of mixing of fines were found to influence the FPF with significantly different device fractions. FPF of AMK LDPI formulations using Rotahaler as the delivery device at 30, 60, and 90 L/min were found to be 21.85% +/- 2.2% and 24.6% +/- 2.4%, 25.9% +/- 1.8% and 29.2% +/- 2.1%, and 29.5% +/- 2.6% and 34.2% +/- 2.0% for AMK1 and AMK2, respectively. From the studies performed in this investigation, it was observed that liposomal charge, addition of fines and order of mixing fines, has a significant effect (P < .05) on in vitro deposition of drug from LDPI formulation.

Development of Liposomal Amphotericin B Dry Powder Inhaler Formulation

The purpose of our study was to prepare and optimize liposomal Amphotericin B (AMB) dry powder inhaler (DPI) formulation for treatment of invasive lung fungal infection. Liposomes were prepared by reverse phase evaporation technique using ethyl acetate and ethanol (1:1) as organic solvents to avoid a possible risk for human health and to impart adequate stability of the vesicles. Drug lipid ratio was 1:10 with membrane composition of hydrogenated soyaphosphatidylcholine; cholesterol and either saturated soyaphosphatidylglycerol (7:3:0.5) or stearylamine (1:1:0.1) was used to prepare negatively (AMB1) and positively (AMB2) charged liposomes, respectively. Liposomes were extruded through 2 μm polycarbonate membrane, separated from unentrapped drug and subjected to lyophilization using Tris buffer containing cryoprotectants in various mass ratios. Sucrose was found to be the best cryoprotectant for liposomal AMB in a mass ratio of lipid: sucrose at 1:5 for AMB1 and AMB2, respectively. Sorbolac 400 and sieved Pharmatose 325 M (500#) in varying mass ratios were used as carriers to prepare the liposomal DPI formulations and subjected to determination of angle of repose, compressibility index, dispersibility index, water content, scanning electron microscopy, and fine particle fraction (FPF). Carrier blend of Sorbolac 400 and 10% sieved Pharmatose 325 M (liposome: carrier ratio to be 1:6) resulted in 22.6 ± 2.2% and 16.8 ± 2.2% FPF for AMB1 and AMB2, respectively with significantly different (p >. 05) device fraction. Percent dug retention studies were conducted at different storage conditions and demonstrated a shelf life over 1 year at refrigerated storage condition (2–8°C).

Liposomes Containing Distamycins: Preparation, Characterization and Antiproliferative Activity

This article describes the production and characterization of two liposome formulations containing antitumor drugs, namely distamycin A (Dist) and a new alkyl derivative of distamycin A (C16-Dist). Egg-PC/cholesterol liposomes (4:1 mol/mol) were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. The encapsulation efficiency was found to be almost complete for C16-Dist (99.8%), while native distamycin A showed a lower yield (19.0%). The in vitro antiproliferative activity of the distamycins-containing liposomes determined on human leukaemic K562 cells, was 11-fold and 8-fold higher for native and alkyl derivative distamycin A, respectively, compared with that of the corresponding free drugs. Liposomal formulations show an increase in the activity and specificity of distamycins in experimental antitumor therapy.

Stratum corneum lipids liposomes for the topical delivery of 5-aminolevulinic acid in photodynamic therapy of skin cancer: preparation and in vitro permeation study.

BackgroundPhotodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) is a skin cancer therapy that still has limitations due to the low penetration of this drug into the skin. We have proposed in this work a delivery system for 5-ALA based on liposomes having lipid composition similar to the mammalian stratum corneum (SCLLs) in order to optimize its skin delivery in Photodynamic Therapy (PDT) of skin cancers.MethodsSCLLs were obtained by reverse phase evaporation technique and size distribution of the vesicles was determinated by photon correlation spectroscopy. In vitro permeation profile was characterized using hairless mouse skin mounted in modified Franz diffusion cell.ResultsSize exclusion chromatography on gel filtration confirmed vesicle formation. SCLLs obtained by presented a degree of encapsulation of 5-ALA around 5.7%. A distribution of vesicle size centering at around 500 nm and 400 nm respectively for SCLLs and SCLLs containing 5-ALA was found. In vitro 5-ALA permeation study showed that SCLLs preparations presented higher skin retention significantly (p < 0.05) on the epidermis without SC + dermis, with a decreasing of skin permeation compared to aqueous solution.ConclusionsThe in vitro delivery performance provided by SCLLs lead to consider this systems adequate for the 5-ALA-PDT of skin cancer, since SCLLs have delivered 5-ALA to the target skin layers (viable epidermis + dermis) to be treated by topical PDT of skin cancer.

A simple method for preparation of immuno-magnetic liposomes

A simple and readily manoeuverable method for preparing immuno-magnetic liposomes that indigenously contain binding sites for attaching other molecules like antibodies on their exterior surface is described. In this method magnetic unilamellar vesicles are prepared from a mixture of phosphatidylcholine, cholesterol, small amounts of a linear chain aldehyde and colloidal particles of magnetic iron oxide, using a reverse phase evaporation technique. The aldehyde (dedecanal) molecules align themselves among the lipid molecules in the bilayer with their aldehyde groups exposed to the aqueous phase, allowing straight attachment of antibody molecules (human-antimouse IgG-FITC in this case) in one single step. The success of this approach is confirmed by fluorescence microscopy as well as binding of the resulting immuno-magnetic liposomes to their corresponding target cells.

Production and characterization of sulforhodamine B containing large unilamellar vesicles labeled with atrazine

As an essential part in the development of an immunological based dipstick test for screening the presence of hormone disrupting pseudo-estrogens in aqueous samples, liposomes containing sulforhodamine B and labeled with atrazine as a model compound were produced. The hapten–lipid conjugate (atrazine-dipalmitoyl phosphatidyl ethano-lamine, DPPE) is obtained by coupling atrazine to DPPE with a C6 spacer in between. Both the atrazine-C6 spacer intermediate as well as the final reaction product are characterized by TLC, HPLC, GC-MS and 1H-NMR, respectively. The production of the liposomes is based on the reverse-phase evaporation technique. Each individual step of the production is optimized, e.g. the manner of addition of the phases, the duration of sonication and evaporation, the type of recipient, the duration of vortex-mixing and final evaporation, the number of consecutive filtrations, the composition of the dialysis buffer, the ultracentrifugation speed as well as the type of antioxidant used. The obtained liposomes are characterized by electron microscopy and by dynamic light scattering (diameter and size-distribution). In addition, we also determined the lipids, phospholipids, cholesterol, the percentage of oxidized lipids, the amount of encapsulated dye and the quantity of liposomes produced. Ten separate productions spread over different days and following the described optimized procedure yielded a reproducible production of large unilamellar vesicles (diameter: 654 ± 109 nm). Additional characteristics of one single production include: 3.9 × 10 11 liposomes/ml; 2.4 × 10 6 molecules of dye inside; a calculated amount of 1.85 × 10 4 molecules of atrazine outside.

Preparation of liposomes by reverse-phase evaporation using alternative organic solvents

The organic solvents employed in liposome preparation, such as chlorinated solvents, diethyl ether or methanol, although usually removed by evaporation, may remain as traces in the final formulation representing a possible risk for human health and influencing the stability of the vesicles. In order to solve the above mentioned disadvantages, this paper describes the use of different organic solvents, namely ethanol, ethyl acetate and two mixtures of ethanol/ethyl acetate, for the production of liposomes by the reverse phase evaporation technique. After preparation, liposomes were extruded through polycarbonate filters and then characterized by size and encapsulation efficacy. As model drugs retinyl acetate and sodium cromoglycate have been used. The association yield for the hydrophilic drug and the encapsulation yield for the hydrophilic model drug were found, in all the liposome preparations, to be satisfactory. In particular, the use of more polar organic solvents (with respect to diethyl ether) appear to enhance the encapsulation of the hydrophilic drug, sodium cromoglycate. The use of alternative organic solvents todiethyl ether can be proposed with the aim of reducing the toxic problems associated with the presence of residual traces of organic solvents in the final liposome formulation.

Production and antiproliferative activity of liposomes containing the antitumour drug chromomycin A3

In the present paper the production and characterization of liposomes are described as a specialized drug delivery system for chromomycin. Liposomes were prepared by the reverse phase evaporation technique followed by extrusion through polycarbonate filters; afterwards the vesicles were characterized in terms of dimensions, morphology and encapsulation efficacy. The aim of this work was to produce a drug delivery system able to reduce the toxicity problems related to the administration of this drug. The analysis of the in vitro antiproliferative activity on cultured human leukemic K562 cells demonstrated that ionic and neutral liposomes containing chromomycin are 1.5 and 7-fold more effective respectively as compared to the free drug. Based on these results and taking into account the increased solubility of the drug in this system, liposomes could represent a promising drug delivery system for use in the experimental therapy using chromomycin.

Characterization of Drug Release from Liposomal Formulations in Ocular Fluid

The successful application of liposomes in topical ophthalmic drug delivery requires knowledge of vesicle stabilization in the presence of tear fluid. The release of procaine hydrochloride (PCH) from large unilamellar liposomes in the presence of simulated tear fluid was studied in vitro as a function of bilayer lipid content and tear protein composition. Reverse-phase evaporation vesicles were prepared from egg phosphatidylcholine, stearylamine or dicetyl phosphate, and cholesterol. The relationship between lipid composition and encapsulation efficiency, vesicle size, drug leakage upon storage at 4°C, and the release of PCH-loaded liposomes was studied. The encapsulation efficiency was found to be dependent upon the lipid composition used in the liposome preparation. In particular, phosphatidylcholine vesicles containing cholesterol and/or charged lipids had a lower entrapment efficiency than liposomes prepared with phosphatidylcholine alone. However, the drug release rate was reduced significantly by inclusion of cholesterol and/or charged lipids in the liposomes. The release kinetics of the entrapped agent seemed to be a biphasic process and the drug-release in both simulated tear fluid (STF) and pH 7.4 phosphate buffered saline (PBS) solutions followed pseudo first-order kinetics in the early stage of the release profile. The drug-release appeared to be diffusion and/or partition controlled. Drug release from liposomes into STF, pH 7.4 PBS, and five different modified tear formulations was also evaluated. While serum-induced leakage is attributed to high-density lipoprotein-mediated destabilization, it was determined that lactoferrin might be the protein component in tear fluid that has the primary influence on the liposome-entrapped drug release rate.Five local anesthetics, benoxinate, proparacaine, procaine, tetracaine, and benzocaine were entrapped in liposomal vesicles by a reverse-phase evaporation (REV) technique. The release of these structurally similar topical anesthetics entrapped in positively charged liposomes (egg phosphatidylcholine, stearylamine, and cholesterol in a 7:2:1 molar ratio) was evaluated in a simulated tear fluid and pH 7.4 phosphate buffered saline solution. The liposomes appeared to be useful carriers for these drugs to retard their in vitro release in tear fluid and perhaps sustain or control their release in the eye for better therapeutic efficacy. An analysis of the release data demonstrated that for this series of drugs, drug partition coefficient has the largest effect on release rate, with molecular weight exhibiting a smaller effect. Release rate was found to decrease with increased lipophilicity or increased molecular weight.

Formulation study for the antitumor drug camptothecin: liposomes, micellar solutions and a microemulsion

In the present paper we describe the production and characterization of specialized delivery systems for camptothecin, namely liposomes, micellar solutions and microemulsion. For instance, liposomes were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. Liposomes were characterized in term of dimensions, morphology and encapsulation efficacy. All the formulations were designed firstly to increase the solubility of camptothecin in aqueous environment and secondly to reduce the toxicity problems related to the administration of this drug. The analysis of their in vitro antiproliferative activity on cultured human leukemic K562 cells demonstrated that liposomes, micellar solutions and microemulsion containing camptothecin exert similar or slightly enhanced effect as compared to that shown by the free drug. Based on these results, the specialized delivery systems here proposed typify an interesting starting point for a future use in experimental therapy.

Liposome mediated delivery of water soluble antibiotics to the larvae of aquatic animals

Large phospholipid vesicles loaded with the water soluble antibiotic oxytetracycline were formed using the reverse phase evaporation technique. Addition of cholesterol in the lipid phase and suspension of the liposomes in low pH solutions, greatly improved stability of the liposomes upon storage. Liposomised oxytetracycline was administered to Anemia franciscana nauplii using the bioencapsulation technique. Survival and larval development of the nauplii was not affected by the presence of liposomes in the enrichment medium. Determination of oxytetracycline levels in the liposomal preparations and in the nauplii, using reverse phase high performance liquid chromatography, showed significant amounts of antibiotic in the nauplii. The uptake of the liposomised antibiotic by the nauplii depended on the amount of the administered antibiotic and the duration of the enrichment period.

Intracellular accumulation of ofloxacin-loaded liposomes in human synovial fibroblasts

In order to incorporate ofloxacin within liposomes, the reverse-phase evaporation technique was carried out. The liposome lipid matrix consisted of dipalmitoylphosphatidylcholine-cholesterol-dihexadecylphosphate (4: 3:4 molar ratio). The liposome formulation presented a mean size of 185 +/- 31 nm and had an encapsulation capacity of 5.3 microliters/mumol. The liposome formulation was able to deliver ofloxacin into McCoy cells in a greater amount (2.6-fold) than the free drug, improving antibiotic accumulation.

Liposome-associated retinoids: production, characterization and antiproliferative activity on neoplastic cells

The administration of retinoids has been demonstrated to be of potential utility in the therapy of a wide spectrum of neoplastic pathologies. This is due to the strong antiproliferative effect on neoplastic cells and to the ability to induce differentiation in a large variety of cell lines. In the present paper we describe the preparation and characterization of liposome-associated natural and synthetic retinoids. Liposomes were prepared by reverse phase evaporation technique followed by extrusion through polycarbonate filters. Analysis of the in vitro antiproliferative activity on cultured human leukemic K562 cells demonstrated that liposome-associated retinoids are up to 200 times more active than the corresponding retinoids administered in the free form. Based on these promising results, a proposal is made for in vivo administration of these antiproliferative agents by using liposomes as a drug delivery system.

Effect of Niosome Encapsulation of Methotrexate, Macrophage Activation on Tissue Distribution of Methotrexate and Tumor Size

AbstractMethotrexate was encapsulated in “niosomes” of sorbitan monostearate (Span 60) by the reverse phase evaporation technique. After i.v. infusion into mice methotrexate concentration was higher in all tissues except kidney with niosomal methotrexate than with methotrexate solution. Niosome encapsulation and macrophage activation enhanced the delivery of methotrexate to the tumor and increased its antitumor activity.

Effect of macrophage activation on plasma disposition of niosomal 3H-methotrexate in sarcoma-180 bearing mice.

Methotrexate niosomes were formed by reverse phase evaporation technique using sorbitan monostearate (Span 60) surfactant. The pharmacokinetic parameters of methotrexate (MTX) after macrophage activation via muramyl dipeptide gelatin conjugate were studied and the results compared with those obtained after free MTX and MTX niosomes without macrophage activation. In mice MTX concentrations and tumour regression were higher after macrophage activation.