Reverse-phase Evaporation Vesicles Research Articles

Effect of Formulation Method, Lipid Composition, and PEGylation on Vesicle Lamellarity: A Small-Angle Neutron Scattering Study.

Liposomes are well-established systems for drug delivery and biosensing applications. The design of a liposomal carrier requires careful choice of lipid composition and formulation method. These determine many vesicle properties including lamellarity, which can have a strong effect on both encapsulation efficiency and the efflux rate of encapsulated active compounds. Despite this, a comprehensive study on how the lipid composition and formulation method affect vesicle lamellarity is still lacking. Here, we combine small-angle neutron scattering and cryogenic transmission electron microscopy to study the effect of three different well-established formulation methods followed by extrusion through 100 nm polycarbonate membranes on the resulting vesicle membrane structure. Specifically, we examine vesicles formulated from the commonly used phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) via film hydration followed by (i) agitation on a shaker or (ii) freeze–thawing, or (iii) the reverse-phase evaporation vesicle method. After extrusion, up to half of the total lipid content is still assembled into multilamellar structures. However, we achieved unilamellar vesicle populations when as little as 0.1 mol % PEG-modified lipid was included in the vesicle formulation. Interestingly, DPPC with 5 mol % PEGylated lipid produces a combination of cylindrical micelles and vesicles. In conclusion, our results provide important insights into the effect of the formulation method and lipid composition on producing liposomes with a defined membrane structure.

Ocular Dorzolamide Nanoliposomes for Prolonged IOP Reduction: in-vitroand in-vivo Evaluation in Rabbits.

Dorzolamide ophthalmic drop is one of the most common glaucoma medications but it has a short residence time in the eye. The aim of this study is to develop ocular dorzolamide HCl nanoliposomes (DRZ - nanoliposomes) and to evaluate their potential use for the treatment of ocular hypertension. Nanoliposomes were prepared using Reverse-phase evaporation vesicle (REV) and thin layer hydration (TLH) method with 7:3 and 7:4 molar ratios of phosphatidylcholine:cholesterol. The physicochemical properties of the formulations were investigated. Formulations with 7:4 lipid ratios were evaluated in terms of drug release, physical stability and ex-vivo permeation through the excised albino rabbit cornea. The rabbits in groups of 6 were treated with selected DRZ - nanoliposomes or dorzolamide solution or marketed dorzolamid preparation (Biosopt®) and intraocular pressure (IOP) was monitored. Formulations with 7:4 molar ratio entrapped greater amount of drug compared to those with 7:3 lipid components ratio. DRZ - nanoliposomes with 7:4 lipid ratio showed more transcorneal permeation than Dorzolamide solution (p<0.05); and the formulation prepared by TLH method exhibited higher permeability than that prepared by REV method (p<0.05). The selected DRZ - nanoliposomes showed greater IOP lowering activity and a more prolonged effect compared to dorzolamide solution and Biosopt®. DRZ - nanoliposomes prepared by TLH method with 7:4 ratios showed promising results as a candidate for the treatment of ocular hypertension.

Repositioning of zoledronic acid for breast cancer using transferrin-conjugated liposome

Zoledronic acid (ZOL) is one of the most potent nitrogen containing bisphosphonates (N-BP) which are used for cancer-induced skeletal disease by inhibiting osteoclast-mediated bone resorption. It acts by inhibiting the farnesyldiphosphate synthase which is one of several key enzymes in the mevalonate pathway consequently leading to osteoclast apoptosis. From recent studies, direct anti-cancer effects of bisphosphonates, particularly ZOL, was demonstrated in breast cancers. The synergic anti-cancer effects of ZOL treated with other anti-cancer agents in breast cancer cells were also reported. However, pharmacokinetic properties of ZOL have limited its cytotoxic activities because almost 60 % of injected ZOL are disposed into bone and 40 % of the dose cleared rapidly from blood, so that not enough dose of ZOL delivered to the cancer cells. For this reason, we introduced transferrin-conjugated long-circulating liposomes as targeted delivery system for enhancing the anti-cancer effect of ZOL. Transferrin receptors have been explored as a target to deliver therapeutics into cancer cells due to their increased expression on malignant cells. Liposomes were prepared by a reverse-phase evaporation vesicle method with aqueous ZOL solution. Transferrins were attached to the neutral liposomes via free thiols (–SH) to the PDP-end of the pyridylthiopropionoylamino-PEG-distearoyl-phosphatidyethanolamine (PDP-PEG-DSPE). Liposomes were characterized by measuring size distribution and zeta potential. The mean diameter of liposomes was 190 nm, and the encapsulation efficiency of ZOL was about 10 %, which was determined by HPLC analysis. Based on the 50 % inhibitory concentration (IC50) of ZOL against breast cancer cell MDA-MB-231, increased anti-cancer effect of transferrin-conjugated liposomal ZOL was evaluated compared to free ZOL and non-conjugated liposomal ZOL. In conclusion, transferrin-conjugated liposome can enhance the anti-cancer effect of ZOL and it is suggested that ZOL can be used as an anti-cancer agent with this formulation in breast cancer patients.

Bisphosphonate‐decorated lipid nanoparticles designed as drug carriers for bone diseases

A conjugate of distearoylphosphoethanolamine-polyethylene glycol with 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (thiolBP) was synthesized and incorporated into micelles and liposomes to create mineral-binding nanocarriers for therapeutic agents. The micelles and liposomes were used to encapsulate the anticancer drug doxorubicin (DOX) and a model protein lysozyme (LYZ) by using lipid film hydration (LFH) and reverse-phase evaporation vesicle (REV) methods. The results indicated that the micelles and LFH-derived liposomes were better at DOX loading than the REV-derived liposomes, while the REV method was preferable for encapsulating LYZ. The affinity of the micellar and liposomal formulations to hydroxyapatite (HA) was assessed in vitro, and the results indicated that all the thiolBP-incorporated nanocarriers had stronger HA affinity than their counterparts without thiolBP. The thiolBP-decorated liposomes also displayed a strong binding to a collagen/HA composite scaffold in vitro. More importantly, thiolBP-decorated liposomes gave increased retention in the collagen/HA scaffolds after subcutaneously implantation in rats. The designed liposomes were able to entrap the bone morphogenetic protein-2 in a bioactive form, indicating that the proposed nanocarriers could deliver bioactive factors locally in mineralized scaffolds for bone tissue engineering.

Study of ocular drug delivery system using drug-loaded liposomes

Background:This project aims to formulate and characterize a drug-eluting contact lens to provide controlled release of drug for a longer period of time, using liposomes as drug delivery system.Materials and Methods:Drug delivering contact lenses were created by coating disposable soft contact lenses with ciprofloxacin entrapped in the liposomes. Reverse phase evaporation and lipid film hydration methods were used for the preparation of ciprofloxacin trapping reverse phase evaporation vesicles, that is, unilamellar vesicles (REVs) and multilamellar vesicles (MLVs), respectively. Soya lecithin and cholesterol (CH) were used in the molar ratios of 7:4 and 7:2. The spherical structure of the liposomes, the mean diameter, and their purity were determined by photomicroscopic, transmission electron microscope, and chromatographic analysis, respectively. The prepared liposomes were evaluated for their entrapment efficiency, in vitro drug release, stability, and toxicity.Results:MLVs were larger than REVs with their mean diameter 338.32 nm and also entrapped greater amount of ciprofloxacin. Drug loading and its release from the liposomal vesicles was dependent on CH content. Ciprofloxacin released from the liposomes coated on the contact lenses not only inhibited both Staphylococcus aureus and Pseudomonas aeruginosa on an agar plate but also showed an enhanced antibacterial effect as determined by minimal inhibitory concentrations. Approximately 40% of ciprofloxacin was retained up to a period of 3 months at 4°C. Furthermore, the formulation was found to be nontoxic and also a reduction in toxicity of ciprofloxacin was observed after entrapment when assessed by lymphocyte toxicity assay and chick embryo inoculation.Conclusions:An innovative drug delivery system consisting of drug-loaded liposomes coated onto the surface of contact lenses has been developed. This system is highly specific in terms of localized and sustained application of the drug.

Diphtheria toxoid conformation in the context of its nanoencapsulation within liposomal particles sandwiched by chitosan

Chitosan (α-(1-4)-amino-2-deoxy-β-D-glucan) is a deacetylated form of chitin, a polysaccharide from crustacean shells. Its unique characteristics, such as positive charge, biodegradability, biocompatibility, nontoxicity, and rigid structure, make this macromolecule ideal for an oral vaccine delivery system. We prepared reverse-phase evaporation vesicles (REVs) sandwiched by chitosan (Chi) and polyvinylic alcohol (PVA). However, in this method, there are still some problems to be circumvented related to protein stabilization. During the inverted micelle phase of protein nanoencapsulation, hydrophobic interfaces are expanded, leading to interfacial adsorption, followed by protein unfolding and aggregation. Here, spectroscopic and immunological techniques were used to ascertain the effects of the Hoffmeister series ions on diphtheria toxoid (Dtxd) stability during the inverted micelle phase. A correlation was established between the salts used in aqueous solutions and the changes in Dtxd solubility and conformation. Dtxd α-helical content was quite stable, which led us to conclude that encapsulation occurred without protein aggregation or without exposition of hydrophobic residues. Dtxd aggregation was 98% avoided by the kosmotropic, PO2–4. This ion was used to prepare a stable Dtxd and immunologically recognized REV-Chi-PVA formulation in the presence of 50 mM of PO2–4. Under these conditions, the Dtxd retained its immunological identity. Therefore, we could obtain the maximum Dtxd solubility and stability after contact with CH3CO2C2H5 to begin its nanoencapsulation within ideal conditions. This was a technological breakthrough, because a simple solution, such as salt, addition avoided heterologous protein use.

Treatment of Experimental Autoimmune Uveoretinitis with Intravitreal Injection of Tacrolimus (FK506) Encapsulated in Liposomes

PURPOSE. To evaluate the effects of intravitreal injection of liposomes encapsulating tacrolimus (FK506) on experimental autoimmune uveoretinitis (EAU) in Lewis rats. METHODS. Liposomes containing tacrolimus were prepared by reverse-phase evaporation vesicles. EAU was induced in Lewis rats by subcutaneous injection of interphotoreceptor retinoid-binding protein R16 peptide emulsified in adjuvant. Ten days later, rats were intravitreally injected with saline, tacrolimus, tacrolimus-loaded liposomes, or unloaded liposomes. Clinical signs of inflammation and ocular histologic sections were observed and graded. Retinal function was evaluated by electroretinography (ERG). Tacrolimus concentration was determined in the vitreous body and serum by ELISA. Ocular biodistribution of rhodamine-conjugated liposomes containing tacrolimus (tacrolimus-Rh-lip) was analyzed with a laser scanning confocal microscope. To evaluate the systemic effect of intravitreally injected tacrolimus, delayed-type hypersensitivity (DTH) and lymphocyte proliferation assay (LPA) responses were detected. RESULTS. Treatment of EAU with intravitreal injection of liposomal tacrolimus significantly reduced intraocular inflammation and markedly inhibited the development of EAU, as determined in clinical and histopathologic analyses. No toxic effects could be detected as evaluated by ERG. The concentration of tacrolimus in ocular fluids remained for as long as 14 days after liposomal injection of tacrolimus. Confocal microscopy showed a transretinal distribution of the liposomal particles. DTH and LPA responses were not impaired in liposomal tacrolimus-treated rats. CONCLUSIONS. Intravitreal injection of liposomal tacrolimus was highly effective in suppressing the process of EAU without any side effects on retinal function or systemic cellular immunity. This treatment may represent a new option for the management of intraocular inflammation.

Preparation and Evaluation of Thermosensitive Liposomal Hydrogel for Enhanced Transcorneal Permeation of Ofloxacin

Ofloxacin, available as ophthalmic solution, has two major problems: first, it needs frequent administration every 4 hours or even every 1 hour to treat severe eye infection; second, there is formation of white crystalline deposit on cornea due to its pH-dependent solubility, which is very low at pH of corneal fluid. In order to provide a solution to previous problems, ofloxacin in this study is prepared as topically effective in situ thermosensitive prolonged release liposomal hydrogel. Two preparation procedures were carried out, leading to the formation of multilamellar vesicles (MLVs) and reverse-phase evaporation vesicles (REVs) at pH 7.4. Effects of method of preparation, lipid content, and charge inducers on encapsulation efficiency were studied. For the preparation of in situ thermosensitive hydrogel, chitosan/beta-glycerophosphate system was synthesized and used as carrier for ofloxacin liposomes. The effect of addition of liposomes on gelation temperature, gelation time, and rheological behaviors of the hydrogel were evaluated. In vitro transcorneal permeation was also determined. MLVs entrapped greater amount of ofloxacin than REVs liposomes at pH 7.4; drug loading was increased by including charge-inducing agent and by increasing cholesterol content until a certain limit. The gelation time was decreased by the addition of liposomes into the hydrogel. The prepared liposomal hydrogel enhances the transcorneal permeation sevenfold more than the aqueous solution. These results suggested that the in situ thermosensitive ofloxacin liposomal hydrogel ensures steady and prolonged transcorneal permeation, which improves the ocular bioavailability, minimizes the need for frequent administration, and decreases the ocular side effect of ofloxacin.

Comparison of liposome based antigen delivery systems for protection against Leishmania donovani

Liposomes have been widely exploited as antigen delivery systems for a variety of diseases including leishmaniasis. These vesicles can be prepared in various ways which may affect the immunogenicity of the encapsulated antigens. In this study we compared the vaccine potentiality of three cationic formulations with Leishmania donovani promastigote membrane antigens (LAg) and the best vesicle was evaluated for long-term protection against experimental visceral leishmaniasis. We immunized mice with LAg encapsulated in multilamellar vesicles (MLV), dehydration–rehydration vesicles (DRV) and reverse-phase evaporation vesicles (REV) and challenged them with parasites ten days after vaccination. LAg in MLV or DRV induced almost complete protection, while LAg alone or entrapped in REV exhibited partial resistance. Protection observed with antigen incorporated MLV or DRV was predominantly Th1 as evidenced by elicitation of significantly high DTH, IgG2a antibodies and IFN-γ. MLV encapsulated LAg demonstrated durable cell-mediated immunity and mice challenged ten weeks after vaccination could also resist experimental challenge strongly. Field trials of L. donovani vaccine were unsatisfactory mainly due to lack of an appropriate adjuvant. Cationic MLV when used as adjuvant with protein antigens induced sustained Th1 immunity. Adjuvant potential of cationic MLV can be utilized to design subunit vaccines.

LUVs Recovered with Chitosan: A New Preparation for Vaccine Delivery

Chitosan, α-(1–4)-amino-2-deoxy-β-D-glucan, is a deacetylated form of chitin, an abundant natural polysaccharide present in crustacean shells. Its unique characteristics such as positive charge, biodegradability, biocompatibility, nontoxicity, and rigid linear molecular structure make this macromolecule ideal as drug carrier. The association between chitosan and liposomes was carefully described, where REVs (reverse phase evaporation vesicles) were sandwiched by chitosan. The usage of these particles in vaccine formulation is here proposed for the first time in the literature. The Chitosan-REVs now stabilized by polyvinilic alcohol were the vehicle for Diphtheria toxoid (Dtxd). Round chitosan-sandwiched REVs (REVs-Chi) particles of 373 ± 17 nm containing 65% Dtxd were obtained. After 200 min of incubation in a simulated gastric fluid, 70% of the Dtxd was liberated from REVs-Chi in comparison to 100% of Dtxd liberated from pure REVs. In PBS, the Dtxd liberation from REVS-Chi was about 60%. Mice were immunized with Dtxd encapsulated within REVs-Chi and with other REVs/Dtxd formulations adsorbed onto Freund adjuvant or alumen [AIF and Al(OH)3]. The response patterns and the immune maturity were measured by IgG1 and IgG2a titrations. REVs-Chi containing Dtxd elicited both antibodies production giving the animals higher immune response and selectivity. It was interesting that the memory of those mice immunized with REVs-Chi containing Dtxd enhanced, after booster, antibody production by 47% in contrast with 17 and 7% in mice immunized with the antigen vehiculated in REVs-AIF or REVs-Al(OH)3, respectively.

UV-induced drug release from photoactive REV sensitized by suprofen

In order to achieve local administration of drugs, calcein (CAL) encapsulated reverse phase evaporation vesicles (REV) carrying photoactive destabilization agent suprofen (SPF) in the lipid bilayer were prepared. Effect of both UV-A and UV-B photoactivation of liposomal membrane incorporated SPF on the destabilization of the liposome bilayer and the release of encapsulated CAL was investigated. Standard REV of phosphatidylcholine (PC):cholesterol (CHOL) in 7:3 molar ratio, and photoactive REV of PC:CHOL:SPF, and DPPC:CHOL:SPF in 7:3:3 molar ratio were prepared. CAL encapsulation efficiency (EE (%)) and in situ release was studied. SPF incorporation in the PC REV membrane led to approximately 5% increase in the EE (34%) in comparison to standard REV (29%). EE decreased (21%) when DPPC was used to replace PC. Exposure to UV-B caused the highest CAL release. The lowest release was from the unexposed REV. DPPC led to a higher liposomal membrane stability (lower CAL release) than PC. A linear relationship was observed between UV-B exposure duration and REV permeability. This study revealed that membrane destabilization of SPF incorporated REV was best achieved upon photoactivation of the membrane-localized SPF by a 40 min exposure to UV-B.

EPR studies of phospholipid bilayers after lipoperoxidation: 1. Inner molecular order and fluidity gradient

The molecular order of fatty acyl chains in oriented lipid bilayers on solid support (SPB), made of either natural or synthetic phospholipids oxidized by Fenton reagent and probed with spin labeled lecithin (5-DSPC) was studied by means of EPR spectrometry. Phospholipids (ASPC, EYPC, mitochondrial extract) were oxidized as either aqueous buffer/methanol dispersions or reverse-phase evaporation vesicles (REV) suspensions. Oxidation was preliminarily revealed both by assaying MDA and by detecting conjugated dienes. Oxidized phospholipid species was quantified by preparative TLC. The degree of order in oriented lipid bilayers of samples containing oxidized phospholipids was estimated by the loss of EPR spectral anisotropy, and an empirical index of the related bilayer disorder was calculated from the second derivative spectra. Bilayers made of each non-oxidized phospholipid species from either ethanol solutions or REV suspensions showed the highest anisotropy, while the increasing presence of oxidized lipids in the samples resulted in progressive loss of EPR spectral anisotropy. In contrast, vesicles containing 40% of the oxidized species maintained an unaltered fluidity gradient, while REV formation was hindered by oxidized phospholipid percentages higher than 45% for ASPC and EYPC, and 35% for Mitochondrial lipids (MtL). It is concluded that the early stages of lipoperoxidation bring about disordering of the phospholipid bilayer interior rather than fluidity alterations, and that prolonged oxidation may result in loss of structural and chemical properties of the bilayer until the structure no longer holds.

Release property of temperature-sensitive liposome containing poly( N-isopropylacrylamide)

Multilamellar vesicles(MLVs) of Egg phosphatidylcholine (PC) were prepared by either reverse-phase evaporation or film hydration method, and dioleoylphosphatidylethanolamine (DOPE) liposomes by detergent removal. Their temperature-dependent release behaviours were investigated in the presence of copolymer of N-isopropylacrylamide and octadecylamine (poly(NIPAM-co-ODA)) or copolymer of N-isopropylacrylamide, acrylic acid and octadecylamine (poly(NIPAM-co-AA-co-ODA)) using calcein a fluorescence probe. By a temperature jump from 17 to 40 °C, crossing the transition temperature of poly(NIPAM-co-ODA), ∼29 °C, the release from reverse-phase evaporation vesicles (REVs) containing the polymer in 270 s increased in a saturation manner until the mixing ratio of polymer to lipid was 0.1, where the degree of release was ∼55%. For the film hydration vesicles (FHVs), the release pattern with polymer to lipid ratio was similar to that of REVs, but the degree of release under the same conditions was much less than in case of REVs, even though both are composed of the same phospholipid. Since the bilayers of REVs are less in number, and more closely stacked each other than those of FHVs, the defect in the bilayers of REVs would be induced more easily by a thermal contraction of the polymer. For DOPE liposomes, the degrees of releases under the same conditions as in the REVs and the FHVs of egg PC were >80%. The extensive release is probably because that the DOPE liposomes disintegrated into nonbilayer structure. On the other hand, in the temperature-dependent release from liposomes mixed with either poly(NIPAM-co-ODA) or poly(NIPAM-co-AA-co-ODA), of which the transition temperatures are ∼29 and 37 °C, respectively, REVs and FHVs started to release significantly around the transition temperature of each polymer. However, for DOPE liposomes the temperature where the liposomes start to release is not clear and an appreciable amount of calcein released below the transition temperature. Therefore, REVs and FHVs are expected to be more favorable than DOPE liposomes when the off-on behavior of release at a certain temperature is desired. Of the egg PC liposomes, REVs would be a better choice than FHVs in aspects of degree of release.

High entrapment of insulin and bovine serum albumin into neutral and positively-charged liposomes by the remote loading method.

Remote loading of insulin and bovine serum albumin (BSA) into neutral or positively-charged liposomes by incubation under a transmembrane pH gradient or non-pH gradient was investigated. Trapping efficiencies in several incubation conditions were compared with those of the conventional reverse-phase evaporation vesicle method (c-REV method). For neutral liposomes, insulin could not be effectively loaded into the liposomes by incubation, regardless of the incubation conditions. The trapping efficiency of insulin into positively-charged liposomes was higher than that of neutral liposomes, especially by the pH-gradient method. Insulin could be loaded into positively-charged liposomes about twofold more efficiently than by the pH-gradient method, compared with the c-REV method. Insulin distributed more on the surface of liposomes by the pH-gradient method than by the c-REV method. BSA showed significantly higher affinity to positively-charged liposomes than to neutral ones by various methods. However, the transmembrane pH-gradient method did not increase BSA loading into liposomes, compared with the c-REV and the non-pH-gradient methods. Our results suggest that the pH-gradient method, combining electrostatic interactions, may be useful for preparation of liposomal insulin and that the high hydrophobicity of BSA may not increase the remote loading of BSA into liposomes by the pH-gradient method.

Remote loading of diclofenac, insulin and fluorescein isothiocyanate labeled insulin into liposomes by pH and acetate gradient methods

Remote loading of the model drugs diclofenac, insulin and fluorescein isothiocyanate labeled insulin (FITC-insulin) into liposomes by formation of transmembrane gradients were examined. A trapping efficiency of almost 100% was obtained for liposomal diclofenac, by the calcium acetate gradient method, whereas liposomes prepared by the conventional reverse-phase evaporation vesicle method had 1–8% trapping efficiencies. Soybean-derived sterol was a better stabilizer of the dipalmitoylphosphatidylcholine bilayer membrane than cholesterol, as shown from trapping efficiencies and drug release. The pH gradient method resulted in a 5–50% of FITC-insulin liposomal trapping efficiency, while insulin could not be loaded by this method. Liposomes released calcein in response to insulin, showing insulin interacts with the liposomal membrane in the presence of a transmembrane gradient. The present work has demonstrated a remote loading method for weak acids such as diclofenac into liposomes by the acetate gradient method. From the result of remote loading of FITC-insulin into liposomes by the pH gradient method, this method may be available for the preparation of liposomal peptides.

Temperature-sensitive releases from liposomes containing hydrophobically modified poly(N-isopropylacrylamide)

Novel temperature-sensitive Liposomes containing hydrophobically modified poly(N-isopropylacrylamide) (HPNIPAM) and their release behaviors were investigated using calcein as a fluorescence probe. Above the lower critical solution temperature (LCST) of the polymer (e.g., 40‡C), the degree of calcein release in 280 sec from reverse-phase evaporation vesicles (REVs) of egg phosphatidylcholine (egg PC) was 43 %, while egg PC MLVs was 16 %. Such a large difference of release may be attributed to the lamellarity of Liposomes. The incorporation of dioleoyl-phosphatidylethanolamine (DOPE) into the PC bilayer enhanced the release by 10–13 % at 40‡C, probably due to the increased instability of mixture bilayers. Meanwhile, a temperature-sensitive device of DOPE Liposomes was prepared by using HPNIPAM as a stabilizer. The optimal ratio of HPNIPAM to lipid to stabilize the bilayer was 0.1. Above the LCST (e.g., 40‡C), the release percentage was about 80% of the entrapped calcein. DOPE Liposomes were the most temperature-sensitive among Liposomes tested. This is probably because DOPE Liposomes disintegrate into a non-liposomal phase, such as hexagonal (H£), by a thermal contraction of HPNIPAM.

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.

ｐＨ勾配法によるニムスチン封入リポソームの調製と評価

Nimustine (ACNU), one of water-soluble nitrosoureas, is used for treatment of some types of tumors. Since this drug can penetrate through the blood-brain barrier, it has been used especially for the treatment of primary brain tumors. ACNU is, however, decomposed rapidly in the blood or cerebrospinal fluid (CSF). In order to overcome this short-coming, we attempted to encapsulate ACNU in liposomes. Liposomes composed of egg phosphatidylcholine were prepared according to the procedure of the reverse-phase evaporation vesicle (REV) method. ACNU could be encapsulated efficiently into liposomes using a transmembrane pH gradient. Encapsulation efficiencies of ACNU with this method were shown to be more than 80%. The mean diameter of liposomes encapsulating ACNU (ACNU liposome) was about 300 nm. These liposomes were stable for more than 7 days at 4°C in storage. When 1 ml of ACNU solution(1 mg/ml) was added to 15 ml of artificial CSF at 37°C, concentrations of ACNU in the solution were 0.28 μg/ml at 3 hr after addition, and no ACNU was detected at 6 hr after addition. On the other hand, when 1 ml of ACNU liposome(1 mg/ml) was added to artificial CSF, concentrations of ACNU in the solution and liposomes were 6.71 and 2.62 μg/ml, respectively at 3 hr after addition. ACNU was detected at 9 hr in artificial CSF and liposomes. These results suggest that the pH gradient method is useful for encapsulation of ACNU in liposomes and the liposome formulation is useful for stabilization of ACNU in artificial CSF.

Reconstitution of beef heart mitochondrial F 0F 1 in reverse phase evaporation vesicles

Beef heart mitochondrial F 0F 1 was reconstituted in proteoliposomes by a new procedure. MF 0F 1 was inserted in preformed reverse phase evaporation vesicles of large diameters prepared from asolectin (MF 0F 1–REV). Reconstitution was mediated by Triton X-100, which was subsequently removed by treatment with Bio-Beads. Parameters which resulted in optimal reconstitution were described. The MF 0F 1–REV proteoliposomes catalyzed an exchange between P i and ATP and were capable of proton pumping. Both reactions were inhibited by oligomycin and uncoupler of oxidative phosphorylation. The range of P i–ATP exchange activity of the proteoliposomes (70–110 nmol min −1 mg −1) compared favorably with activities obtained in vesicles reconstituted by cholate dialysis or cholate dilution. The most important aspect of this method is that, unlike other reconstitution methods, exogenous F 1 and other coupling factors are not required to obtain high P i–ATP exchange activity by MF 0F 1–REV. This simple and rapid reconstitution procedure should be useful for future studies dealing with functional analysis of MF 0F 1.

Encapsulation of native crotoxin in liposomes: A safe approach for the production of antivenom and vaccination against Crotalus durissus terrificus venom

Crotoxin, the neurotoxic component of Crotalus durissus terrificus (Cdt) venom that displays phospholipase A 2 activity, was successfully encapsulated into dehydration-rehydration vesicles (DRV/crotoxin) and reverse-phase evaporation vesicles (REV/crotoxin) made from sphingomyelin and cholesterol. The encapsulation efficiency of native crotoxin was higher in DRV/crotoxin than in REV/crotoxin. DRV/crotoxin was not toxic when i.v. inoculated in mice at a dose of crotoxin as high as 91 times its ld 50 or when s.c. inoculated at 42 times its ld 50. On the other hand, crotoxin released from DRV/crotoxin retained its original toxicity. REV/crotoxin was found to be at least 1.9 times more toxic than DRV/crotoxin. The fact that DRV/crotoxin retained crotoxin more efficiently than REV/crotoxin may account for the difference in acute toxicity between the two preparations. DRV/crotoxin, when s.c. inoculated in mice, induced anti-crotoxin antibodies that protected animals against the lethal effect of Cdt venom. Following immunization with three doses of DRV/crotoxin (3 × 20 μg of crotoxin/mouse) and challenge with 8 × ld 50 of Cdt venom, 75% of mice were protected. The DRV/crotoxin preparation was compared to crotoxin emulsified in Freund's adjuvant (FCA/crotoxin). DRV/crotoxin was found to be less toxic than FCA/crotoxin, and to induce lower levels of anti-crotoxin antibodies but similar levels of protection when inoculated at high doses (20 or 70 μg crotoxin/mouse). When DRV/crotoxin was adsorbed to alum at the time of immunization, it induced antibody and protection levels comparable to those produced by FCA/crotoxin.