Effects of Triton X-100 concentration and incubation temperature on carboxyfluorescein release from multilamellar liposomes

Carboxyfluorescein is the most commonly used probe to measure the rate of release of vesicle contents. The validity of the data obtained by this method depends on obtaining an end point based on the complete release of the dye on treatment of the liposomes with a detergent, usually Triton X-100. However, Triton does not completely release entrapped carboxyfluorescein from multilamellar liposomes and the amount and rate of release of marker upon detergent treatment is a function of lipid composition of the liposome, Triton concentration and temperature and duration of detergent incubation. The fluorescence `end point' for distearoyl--[alpha]-phosphatidylcholine/cholesterol (2:1, mol%) multilamellar liposomes treated with 0.5% Triton at 22[deg]C (a condition often used) is only about one-fifth the value for liposomes treated with 5% Triton at 72[deg]C. The conditions of treatment appear to affect the release of carboxyfluorescein from the lipid of the partially or completely disrupted liposome and the subsequent partitioning of the free dye into the aqueous phase. This effect can lead to serious errors in the interpretation of multilamellar liposome stability data. However, Triton allows complete release of entrapped dye from small unilamellar vesicles under all conditions tested.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26095/1/0000171.pd

Interactions of neomycin and calcium in synaptosomal membranes and polyphosphoinositide monolayers

Neomycin and related aminoglycosidic antibiotics displace calcium from synaptosomes of guinea pig cerebral cortex and from preparations of phosphatidylinositol diphosphate. At low drug concentrations, inhibition of synaptosomal calcium binding is competitive (Ki = 3[middle dot]10-5M), at high concentrations it is non-competitive (Ki = 4[middle dot]10-4M). Monomolecular films of phosphatidylinositol diphosphate are contracted by low concentrations of neomycin in the subphase, and are expanded at high concentrations. This expansion persists even at the collapse pressure indicating a strong interaction between the drug and the lipid.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21792/1/0000187.pd

Membrane effects of aminoglycoside antibiotics measured in liposomes containing the fluorescent probe, 1-anilino-8-naphthalene sulfonate

The mechanism of membrane disturbance by aminoglycoside antibiotics was investigated in liposomes containing the fluorescent probe, 1-anilino-8-naphthalene sulfonate (ANS). Liposomes of PC and different anionic phospholipids (1:1 to 15:1 molar ratios) were challenged with aminoglycosides in the presence of low (1 [mu]M) and high (3 mM) concentrations of calcium. Liposomes containing PIP2 showed the greatest drug-induced changes in ANS fluorescence in the presence of high and low concentrations of calcium and at all PC:PIP2 molar ratios tested. Liposomes containing other anionic phospholipids (PS, PI and PIP2) were not reactive toward aminoglycosides in the presence of 3 mM calcium or when the ratio of PC to anionic lipid was increased to 10:1. The aminoglycoside-induced changes of ANS flouorescence were not due to any changes in the emission spectrum of ANS, nor to changes in quantum yield, nor to a change in the binding affinity of ANS. It is concluded that a specific aminoglycoside-PIP2 interaction results in phase separation of PC and PIP2 and thus increases the number of available ANS binding sites in PC:PIP2 liposomes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25983/1/0000049.pd

Aminoglycoside antibiotics preferentially increase permeability in phosphoinositide-containing membranes: a study with carboxyfluorescein in liposomes

The rate of release from multilamellar liposomes of the fluoroscent probe carboxyfluorescein was determined as a measure of membrane permeability. Liposomes of phosphatidylcholine and different anionic phospholipids were incubated with low (1 [mu]M) and high (3 mM) concentrations of calcium in the absence or presence of aminoglycoside antibiotics. The leakage of carboxyfluorescein into the medium was not caused by liposomal fusion as no vesicle fusion was observed in experiments with terbium and dipicolinic acid-loaded liposomes. The basal rate of carboxyfluorescein release (in the absence or presence of 1 [mu]M calcium) from all types of liposomes ranged from 0.1 to 0.3% of trapped carboxyfluorescein per hour. The presence of 3 mM calcium caused the greatest increase in the rate of carboxyfluorescein release (about 9-fold) in liposomes containing phosphatidylinositol 4,5-bisphosphate (PIP2) whereas liposomes containing the other anionic phospholipids (phosphatidylserine, phosphatidylinositol and phosphatidylinositol 4-phosphate) showed an approximate 5-fold increase. In the presence of 1 [mu]M calcium, the aminoglycosides neomycin and gentamicin also increased the rate of carboxyfluorescein release, with PIP2-containing liposomes showing a 3-5-times greater response than the other liposomes, releasing up to 4.6% of trapped carboxyfluorescein per hour. This drug-induced release was dose-dependent and antagonized by calcium. In the presence of 3 mM calcium, 0.1 mM gentamicin or neomycin were ineffective while the drug at 1 mM affected carboxyfluorescein release from PIP2-liposomes only. The aminoglycoside antibiotics, neomycin, gentamicin, tobramycin, kanamycin, amikacin, netilmicin, as well as neamine and spectinomycin (all at 0.1 mM) showed a graded effect on the rate of carboxyfluorescein release from PIP2-containing vesicles in the presence of 0.1 mM calcium. The magnitude of the effect correlated well with the ototoxicity of the drugs previously determined directly in cochlear perfusions in the guinea pig. The study demonstrates that aminoglycoside antibiotics are capable of altering membrane permeabilities and that this effect is most pronounced if PIP2 is present in the bilayers. The excellent correlation between this membrane action and the in-situ toxicity of the drugs further establishes the specific role of PIP2 in the molecular mechanism of aminoglycoside-induced hearing loss. Moreover, it confirms the usefulness of such physicochemical models for the screening and prediction of aminoglycoside toxicity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26611/1/0000152.pd

Effect of Ca2+ on thermotropic properties of saturated phosphatidylcholine liposomes

The effect of various concentrations of calcium ion (Ca2+) on dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and mixed DPPC/DSPC (1:1) liposomes was studied by differential scanning calorimetry. Ca2+ concentrations of 10 mM and above split the main transition peak of DPPC into two distinguishable components, and, at 30 mM and above, also resulted in the disappearance of a pre-transition peak. The effect of Ca2+ on DSPC liposomes was even more dramatic in that it induced a more definitive split in the main transition peak and caused the loss of the pretransition peak at only 10 mM concentration. The thermograms of the DPPC/DSPC mixed liposomes were unaltered in the presence of Ca2+, even at a concentration of 50 mM. Whether or not Ca2+ is able to alter thermograms of phosphatidylcholine liposomes appears to be dependent on the degree of molecular order of the bilayer prior to interaction with Ca2+.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24008/1/0000257.pd

Mechanisms of topical delivery of liposomally entrapped drugs

Our research on the mechanism by which liposomally entrapped solutes are transported across the skin was prompted by an investigation reported in the literature which con- cluded with meager supporting evidence that liposomes containing triamcinolone acetonide penetrated the stratum corneum intact and, thereby, increased skin absorption. To elucidate the mechanism we used glucose, hydrocortisone, progesterone and multilamellar DPPC liposomes. Experimental strategies involved: DSC determinations, in vivo permeation of hairless mouse skin by liposomes, by liposome-entrapped solutes (15:1) and by solutes in simple solution; and in vitro release kinetics of liposome-entrapped solutes.The liposomes neither penetrated the skin nor fused with the stratum corneum. Progesterone and hydrocortisone, which were intercalated in the bilayer structure, permeated the skin with ease comparable to free drug. The skin transport of the highly polar glucose entrapped in the aqueous regions of the liposome was markedly slow as compared to the free species. Physical model analysis indicated that the slow release rate of glucose out of the liposome was the rate-determining step as compared to the relatively rapid skin permeation of the free solute. For the hydrophobic progesterone and hydrocortisone, quantitative analyses suggested direct transfer of drug from the liposome to the surface phases of skin and subsequent diffusion through the tissue. Considering this mechanism and owing to increased solubility of lipophilic drugs in liposomes, more total drug may be delivered through the skin by liposomes relative to simple aqueous solution.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25506/1/0000047.pd

Relative uptake of minoxidil into appendages and stratum corneum and permeation through human skin in vitro

We examined uptake of the model therapeutic agent, minoxidil, into appendages, stratum corneum (SC), and through human skin, under the influence of different vehicles. Quantitative estimation of therapeutic drug deposition into all three areas has not previously been reported. Finite doses of minoxidil (2%, w/v) in formulations containing varying amounts of ethanol, propylene glycol (PG), and water (60:20:20, 80:20:0, and 0:80:20 by volume, respectively) were used. Minoxidil in SC (by tape stripping), appendages (by cyanoacrylate casting), and receptor fluid was determined by liquid scintillation counting. At early times (30 min, 2 h), ethanol-containing formulations (60:20:20 and 80:20:0) caused significantly greater minoxidil retention in SC and appendages, compared to the formulation lacking ethanol (0:80:20). A significant increase in minoxidil receptor penetration occurred with the PG-rich 0:80:20 formulation after 12 h. We showed that deposition of minoxidil into appendages, SC, and skin penetration into receptor fluid were similar in magnitude. Transport by the appendageal route is likely to be a key determinant of hair growth promotion by minoxidil. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:712–718, 2010Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/64562/1/21856_ftp.pd

Evaluation of the hairless rat as a model for in vivo percutaneous absorption

Percutaneous absorption of topically applied mannitol and progesterone was compared in vivo with the hairless and hairy rat. Urinary excretion and skin concentration profiles after topical application of mannitol demonstrated that hairless rat skin was a “leakier” barrier to percutaneous absorption of polar compounds than was hairy rat skin, independent of formulation. Liposomal, but not aqueous mannitol was retained in hairy rat skin (> 0.5% after 12 h), whereas only negligible amounts were retained in hairless rat skin, regardless of formulation. Progesterone absorption from hydroalcohol and liposomal formulations into hairless rat skin was about five times greater than that in hairy rat skin. Skin delipidization by acetone resulted in a dramatic reduction in the cutaneous barrier to systemic mannitol absorption, which was much more pronounced in hairy than in hairless rat skin. Histological findings of patulous cysts and enlarged, highly vascularized sebaceous glands in the hairless rat suggested that these structures may enhance polar pathways and provide a lipophilic reservoir relative to the fully developed hair follicles of the hairy rat. Collectively, the results document percutaneous absorption differences as a function of animal model, and also suggest that follicular structures make a major contribution to passive percutaneous absorption.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34489/1/2_ftp.pd

In situ jejunal uptake from liposomal systems

The in situ uptake from the rat jejunum of liposomes prepared from various saturated phosphatidylcholines in the presence or absence of cholesterol was determined. Liposomes prepared from distearoylphosphatidylcholine, with or without cholesterol, were taken up at the fastest rate (apparent first-order uptake of intact liposomes with a half-life of about 15 min). The lipid components of liposomes could not be found in compartments where liposomes would be expected to accumulate, i.e. liver, spleen, lymph nodes, or thoracic lymphatic duct. Furthermore, inulin, when encapsulated in the liposomes, could not be found in the urine. When either PEG-4000 (a non-absorbable marker) or glucose (an absorbable marker) was entrapped within the liposome, all of the liposomes and markers that were taken up still remained associated with the jejunum and the liposomes remained intact.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24965/1/0000392.pd

Topical delivery of liposomally encapsulated gamma-interferon

The extent of uptake of gamma interferon ([gamma]-IFN) in various strata of hairless mouse, human and hamster skin upon application of a liposomal formulation and an aqueous solution were determined by in vitro diffusion cell experiments. For each of the animal species studied, 70-80% of the liposomally entrapped IFN was deposited onto or penetrated into the skin as determined 24 h after in vitro application. However, a significant fraction of this total amount ([approximate] 0.25-0.30) is either adsorbed to or associated with the stratum corneum. The drug content found in the deeper skin strata, where the receptor sites reside, suggests that drug deposition is strongly influenced by the skin species tested. The percent of applied drug found in this strata 24 h after application followed the order: hamster (6.1) >= human (0.9) > hairless mouse (0.3), although the amounts of drug in the total skin of each species tested were approximately the same. This indicates that the deposition of drug into the living epidermis and/or dermis cannot be predicted by determination of the amount of drug in the total skin. The amounts in the deeper skin strata were also in the order of increasing number of follicles/hair in the skin species, suggesting that the transfollicular route is an important pathway for liposomal topical therapeutics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29983/1/0000347.pd