Topical transfection using plasmid DNA in a water-in-oil nanoemulsion.

Expression plasmids encoding chloramphenicol acetyltransferase (CAT) or human interferon-alpha2 cDNA were formulated in water-in-oil nanoemulsions and applied to murine skin. The histological location of transfected cells was assessed by in situ DNA PCR and showed that the deposition of plasmid DNA was primarily in follicular keratinocytes. Transgene expression in the skin was monitored for 24-72 h, following topical application of either single or multiple daily doses by quantitative RT-PCR and ELISA. It was found that transgene expression was optimal at 24 h following topical application of a single dose of water-in-oil nanoemulsion containing plasmid DNA. Dose-response studies using a total dose of 3, 10 or 30 microg of plasmid DNA suggested that topical transfection using nanoemulsions is subject to both threshold and saturation effects. None of the cationic liposome formulations tested as controls mediated transgenic protein expression at levels higher than background values of the ELISAs used to assay transgenic protein. Single and multiple dose experiments using human interferon-alpha2 as a transgene indicated that the efficiency of nanoemulsion mediated transfection was most effective in the context of normal versus atrophic hair follicles. In addition, the total amount of human interferon-alpha2 present in skin appeared to accumulate as a consequence of multiple dosing. Histologic evaluation of treated skin showed no overt signs of toxicity or irritation associated with the short-term application of the nanoemulsions. The results suggest that water-in-oil nanoemulsions can be used to facilitate transfection of follicular keratinocytes in vivo.

Topical transport of hydrophilic compounds using water-in-oil nanoemulsions.

A variety of water-in-oil nanoemulsions were prepared using sorbitan monooleate (Span80), polyoxyethylene 20 sorbitan monooleate (Tween80), olive oil and water. The nanoemulsions were tested for their ability to facilitate transport of a model hydrophilic solute, inulin, across hairless and hairy mouse skin and hairy rat skin following topical in vitro application. The transport of inulin incorporated in water-in-oil nanoemulsions was found to be significantly higher (5- to 15-fold) than that obtained with micellar dispersions or aqueous controls. The rate and extent of inulin transport across hairy mouse skin was found to be highly dependent on the hydrophile-lipophile balance (HLB) of the surfactant mixture in the nanoemulsion. Nanoemuslions prepared using mixtures with lower HLB exhibited significantly higher rate and extent of transport. It was also found that nanoemulsion-mediated transport was independent of molecular size of the hydrophilic solute and the nature of the aqueous phase. More importantly, transport of inulin from nanoemulsions was independent of animal skin characteristics such as stratum corneum thickness and follicle-type. The combined results suggest that water-in-oil nanoemulsions that are compatible with the lipophilic sebum environment of the hair follicle facilitate efficient transport of incorporated hydrophilic solutes and imply that such transport is predominantly transfollicular in nature.

Application of membrane-based dendrimer/DNA complexes for solid phase transfection in vitro and in vivo.

In this study a general description of the use of solid support membranes as the device for DNA delivery mediated by PAMAM dendrimers is presented. In contrast to the other DNA carriers, dendrimer/DNA complexes retain the ability to transfect after drying, which enabled coating or incorporation of complexes into poly(DL-lactide-co-glycolide) or collagen-based bioerodable membranes. These studies provide support for the use of this technology for in vitro and in vivo transfection of skin cells. Expression of luciferase or green fluorescent protein from pCF1-Luc and pEGFP1 plasmids indicated that dendrimer/DNA complexes can mediate transfection after dissociation from the solid support and/or when retained on the surface of the membranes. Modification of the membranes by incorporation of an anionic lipid, phosphatidyl glycerol (PG) at 1-5% concentrations, resulted in more efficient in situ transfection, particularly with dendrimer/DNA complexes formed at the low charge ratios (1-5). We also report data supporting the feasibility of membrane-based dendrimer/DNA complexes, particularly formed at lower than neutralizing conditions, for topical in vivo delivery of DNA to hairless mouse skin.

Perifollicular transgenic expression of human interleukin-1 receptor antagonist protein following topical application of novel liposome-plasmid DNA formulations in vivo.

Expression plasmid DNA for the human interleukin-1 receptor antagonist (IL-1ra) protein was formulated with nonionic:cationic (NC) liposomes or phosphatidylcholine:cationic (PC) liposomes and applied to the auricular skin of hamsters in single- and multiple-dose protocols. Confocal microscopy identified delivery of plasmid DNA proximal to perifollicular cells, and successful transfection of perifollicular cells was identified by immunohistochemistry and ELISA. Skin treated for 3 days with the NC liposomes had statistically significant levels of transgenic IL-1ra present for 5 days post-treatment. Expression of transgenic IL-1ra was specific to areas of skin treated with NC liposomes but not PC liposomes. The results indicate that the NC liposomes can deliver expression plasmid DNA to perifollicular cells and mediate transient transfection in vivo.

Dissolution media for in vitro testing of water-insoluble drugs: effect of surfactant purity and electrolyte on in vitro dissolution of carbamazepine in aqueous solutions of sodium lauryl sulfate.

The intrinsic dissolution rate and solubility of carbamazepine was measured in aqueous solutions of sodium lauryl sulfate (SLS) prepared with two different grades of purity, 95 and 99%, and 95% SLS in 0.15 M NaCl to determine the effect of surface-active impurities and electrolytes. Four significant observations resulted from this work: (1) the equilibrium coefficients calculated from the solubility experiments in the 99% SLS, 95% SLS, and 95% with 0.15 M NaCl SLS solutions were 295, 265, and 233 L/mol, respectively; (2) the dissolution rate enhancement in the 99% SLS was 10% greater than that in the 95% SLS and 95% with 0.15 M NaCl solutions, which were not significantly different; (3) the diffusion coefficients of the drug-loaded micelles estimated from the dissolution experiments were 8.4 x 10(-7) cm2/s for the 99% SLS, 9.5 x 10(-7) cm2/s for the 95% SLS, and 1.2 x 10(-6) cm2/s for the 95% with 0.15 M NaCl; and (4) the critical micelle concentrations for the 99% SLS, 95% SLS, and 95% SLS with 0.15M NaCl were 6.8, 4.2, and 0.35 mM, respectively. The results of this study clearly illustrate the sensitivity of the micelle to impurities and electrolytes with regard to size and loading capacity and the effect these changes have on the solubility and dissolution rate. Therefore, when using surfactants in dissolution media for in vitro testing of dosage forms, consideration must be given to the level of impurities present so that the results are consistent and reliable. Intrinsic dissolution rate, surface tension, or solubility measurements may be useful, convenient methods for identifying changes in the surfactant due to either degradation or lot-to-lot variability.

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.

Penetration of minoxidil from ethanol/propylene glycol solutions: effect of application volume and occlusion.

We have previously established that the relative concentrations of propylene glycol and ethanol as a binary solvent system have a significant effect on the skin penetration of 2% solutions of minoxidil at 50 microL/cm2. The present work extends these studies and investigates the penetration of minoxidil from the different vehicle combinations as functions of application volume and occlusion. Decreasing the application volume has a variable effect which depends on vehicle composition. Penetration of minoxidil from 100% ethanol solutions decreased linearly with application volume. Generally, irrespective of the volume applied, the penetration of minoxidil increased with increasing ethanol fraction with a maximum penetration at 90% ethanol. Penetration from all the formulations was enhanced upon occluding the skin, with greatest increase evident in solutions with higher volatile fraction. Penetration of minoxidil in vivo showed trends similar to those seen in vitro.

Transfollicular drug delivery.

The hair follicle, hair shaft, and sebaceous gland collectively form what is recognized as the pilosebaceous unit. This complex, three-dimensional structure within the skin possesses a unique biochemistry, metabolism and immunology. Recent studies have focused on the hair follicle as a potential pathway for both localized and systemic drug delivery. Greater understanding of the structure and function of the hair follicle may facilitate rational design of drug formulations to target follicular delivery. Targeted drug delivery may enhance current therapeutic approaches to treating diseases of follicular origin. Presented here is a review of follicular drug delivery and a discussion of the feasibility of the pilosebaceous unit as a target site.

The role of rheological properties in mucociliary transport by frog palate ciliated model.

The effect of viscoelastic properties on mucociliary transport rate was investigated using the frog palate ciliated model. Mucociliary transportability of several hydrophilic polymeric gels with widely different viscoelastic characteristics were tested on the frog palate mucociliary model. An apparent negative relationship is observed between the relative transport rate (TR) and storage (G1) or loss (G2) modulus. However, a minimum in relative transport rate is observed at an apparent loss tangent (tan delta) value of between 0.7 and 0.9. A theoretical model for mucociliary transport is presented. The model predicted a minimum in transport rate at tan delta equal to 1.74 after adjustment for primary variation due to storage modulus (G1), which is in agreement with the observed frog palate transport rate. The model isolates the loss tangent (tan delta) and the magnitude of the complex modulus (magnitude of G*) as the important viscoelastic parameters for mucociliary transport. Optimum rheological characteristics with respect to slow transport rate can be achieved by using hydrophilic polymer gels with a large complex modulus and simultaneously with a loss tangent equal to 1.74.

Viscoelastic properties of poly(ethylene oxide) solution.

The viscoelastic properties of poly(ethylene oxide) (PEO) solution were investigated using the dynamic oscillatory testing technique. With this technique, the effect of PEO molecular weight (MW), concentration, composition of mixed solvent systems consisting of propylene glycol, glycerol formal, and water, and the effect of NaCl salt on the viscoelastic properties of PEO solution were determined. Dynamic moduli (G1, G2), magnitude of complex viscosity (magnitude of eta*), and loss tangent (tan delta) were examined over a frequency range of 10(-3)-2.5 Hz at 30 degrees C. The results indicated that low MW PEOs show liquidlike behavior while high elasticity is exhibited by high MW PEOs due to entanglement formation. The complex viscosity, magnitude of eta*, exhibits shear thinning (power-law) characteristics under oscillatory measurements. The relationship between steady shear and complex viscosities follows the Cox-Merz rule over the shear rate and frequency region studied. Both the storage (G1) and loss (G2) modulus increase drastically as the proportion of water in the mixed solvent system increases. Similarly, both G1 and G2 are found to increase while the tan delta decreases with increasing concentration of PEOs. The addition of up to 2% w/w NaCl in an aqueous solution of 10% w/w 2 million MW PEO has no observed detrimental effect on the viscoelastic behavior.

Liposomes as carriers for topical and transdermal delivery.

The delivery of active agents to the skin by liposome carriers is an interdisciplinary topic of great interest today. Data accumulated over the last decade strongly point to important advantages of these drug delivery systems. A symposium devoted to classic and new approaches in the use of liposomal systems was organized and chaired by M. Mezei and E. Touitou as a part of the Jerusalem Conference on Pharmaceutical Sciences and Clinical Pharmacology, held on May 24-30, 1992, in Jerusalem, Israel. The presentations focused on liposomes as tools in the mechanistic study of absorption promoters (T. Nagai), drug liposomal delivery in the skin strata and structures (N. Weiner), interaction of liposomes and niosomes with the human skin (H.E. Junginger), and design and characterization of caffeine liposomal systems for use in hyperproliferative diseases (E. Touitou). Mezei reviewed biodisposition and clinical studies on liposomal dosage forms containing various drugs.

Viscoelasticity of anionic polymers and their mucociliary transport on the frog palate.

The influence of formulation variables on the rheology of polyanionic formulations and the relationships between viscoelastic properties and mucociliary transport rate were investigated. Polymeric samples were oscillated from 0.001 to 5 Hz using either a "cone and plate" or a "coaxial cylinder" measuring system. The mucociliary transport rates of polymeric samples were determined and compared movement of charcoal powder on the frog palate. For the linear polymeric solutions, sodium carboxymethylcellulose and sodium alginate, the elastic modulus (G') increased with increasing amplitudes during frequency scan. However, the G' or viscous modulus (G'') of partially cross-linked polyacrylic acid (cPAA) samples did not change significantly under oscillation. Both G' and G'' of cPAA samples were significantly influenced by the amount of salt present in the formulation. The rheology of 2% (w/w) cPAA in 90:10 (w/w) propylene glycol:alcohol changed from a viscous fluid to a coarse suspension after neutralization. The pH increased gradually when the nonaqueous formulation reacted with water and the maximum dynamic moduli were obtained after incorporating 20% (w/w) water in the formulation. A negative correlation was found between the G' of linear polyanionic samples and the relative transport rate. However, the lowest mucociliary transport rate was observed when the loss tangent (G''/G') was around 0.4-0.5.

Viscoelastic properties of polyacrylic acid gels in mixed solvents.

The objective of this study is to investigate the viscoelastic properties of Carbopol 934P polymeric systems in a variety of mixtures of pharmaceutical solvents. Carbopol 934P neutralized with a 1:1 equivalent ratio of triethanolamine was dissolved in various binary or ternary solvent mixtures consisting of propylene glycol, glycerol formal, and water. Dynamic moduli G' and G'', complex viscosities, eta' and eta'', and loss tangent, tan delta, were examined over a frequency range of 10(-3) to 10 Hz using an oscillatory viscoelastic rheometer at 30 degrees C. The results indicated that for 0.5-1.5 wt% neutralized Carbopol in ternary mixtures, G' and G'' increased by 3-4 orders of magnitude and the phase angle decreased from 80 to 25 degrees when the water content in the solvent mixture increased from 10 to 80 wt%. These studies also indicated that the addition of water to nonaqueous Carbopol 934P polymer systems transforms them from low-viscosity solutions to gels with significant elastic behavior involving physical interaction and entanglement of polymer segments with solvents.

Bulk organic solvent-water systems as a possible model to predict alkyl p-aminobenzoate partitioning in liposomes.

This study compares the bilayer-water distribution coefficients of a homologous series of n-alkyl p-aminobenzoates in liposomes with their respective distribution coefficients in octanol-water, oleyl alcohol-water, and hexane-water systems. The data indicate that the bilayer-water distribution coefficient is quite sensitive to changes in solute structure and to the structural organization of the bilayer and that octanol, oleyl alcohol, and hexane are able to reflect the partitioning changes that occur in the liposomal bilayer with respect to increasing the alkyl chain length of n-alkyl p-aminobenzoates. For this particular homologous series, the hexane-water system tended to underestimate the bilayer-water distribution coefficients, whereas octanol-water and oleyl alcohol-water systems overestimated solute partitioning into the bilayer. The similarity of the lipid environment, with respect to solute partitioning, in the organic solvent system and that in liposomes can be ascertained by using the Collander relationship.

Drug and vehicle deposition from topical applications: use of in vitro mass balance technique with minoxidil solutions.

The disposition of minoxidil and propylene glycol from topical solutions was measured by using an in vitro mass balance technique. The experimental approach included assessment of the following compartments of the skin and the diffusion cell as a function of time: (1) donor compartment; (2) hairless mouse skin surface, epidermis, and dermis; and (3) receiver compartment. Excellent mass balance was achieved for minoxidil at three doses. However, the recovery of propylene glycol depended on both application volume and time. The experiment involving the evaporation of propylene glycol and water from the propylene glycol:ethanol:water (20:60:20, v/v) mixture, which was placed in the well of a tissue culture plate at room temperature and 37 degrees C, substantiated the loss of vehicles to the air. When a thin application of 20 microL/cm2 was used, 60% of the propylene glycol was unaccounted for after 16 h. The evaporation of propylene glycol concentrated the solution to supersaturation, precipitated out the drug, and then stabilized the thermodynamic activity of the drug in the vehicle. The amount of formulation applied influences the rate of concentration and, thus, the time at which minoxidil precipitates. The precipitation limits the amount of minoxidil that can be absorbed and leads to poor percutaneous absorption of drug from the formulation.

Viscometric study of polyacrylic acid systems as mucoadhesive sustained-release gels.

This report describes a novel nonaqueous polymeric formulation that exhibits low-viscosity fluid behavior for ease of spraying with conventional nebulizer, which when sprayed into the nasal cavity, transforms to a high-viscosity gel for efficient retention and drug absorption. The transformation occurs because of the rheological changes induced by a change in the solvent composition of the polymeric formulation in the moist nasal cavity. Such a rheological change would then facilitate enhanced residence time of the drug at the site of administration in order to avoid drainage losses. This study reports the results of the effects of a variety of factors such as solvent composition and polymer concentration on the rheological properties of a polyacrylic acid polymer. An attempt to correlate viscosity enhancement effects with enhanced and sustained-release behavior of propranolol, a drug that undergoes extensive first-pass effects, from such formulations via nasal administration in beagle dogs is also described.

Mixture experimental design in the development of a mucoadhesive gel formulation.

The objective of the present study is to apply response surface methodology to the design and analysis of composite experiments containing independent covariate(s). The approach is illustrated here by the study of viscosity characteristics of a polymeric mucoadhesive formulation in multicomponent solvent vehicles. The nonaqueous formulation will produce a gel network with significant rheological change when in contact with body fluids. The process of water inclusion will induce not only solvent compositional change of the mixture but also concomitant dilution of the polymer concentration. To study the viscosity change over the solvent compositions and polymeric concentrations of interest, an experimental design is utilized consisting of a 10-point simplex-centroid lattice augmented with three interior points at each polymeric concentration. The contour patterns are compared with the experimental data using the variance and lack of fit, starting with the Scheffe linear model and building up to the full cubic model including the covariate terms. The fitted model provides information needed to predict optimum formulations, i.e., initial viscosity of less than 100 cP, but yielding rheological profiles commensurate with high degrees of substantivity when diluted with water. For illustrative purposes, the Carbopol resins neutralized with a 1:1 molar equivalent ratio of triethanolamine in three primary solvents, propylene glycol, glycerol formal, and water, were chosen for this study.

Separation of liposome populations differing in phosphoinositide content by chromatography on immobilized neomycin.

A method is presented by which liposomal populations can be separated based on their content of phosphatidylinositol 4,5-bisphosphate (PIP2). Neomycin is reductively coupled to sepharose 2B and used as the stationary phase in column chromatography. Liposomes of egg lecithin (EL) plus acidic phospholipid containing as little as 10 mol% PIP2 are quantitatively retained in 0.2 M NaCl, while liposomes containing other anionic lipids are recovered to 70-97%. It is possible that lipid redistribution of the bilayers upon contact with the sepharose:neomycin adduct may result in the formatin of multiple populations, and the percent retention by the column for each of the EL:phosphoinositide liposomes would be determined by the new equilibrium established as a result of these interactions. When mixed liposomal populations were chromatographed, the recovery in the 0.2 M NaCl eluates was the sum of the individual recoveries, and there is no significant interchange of lipids between the two liposome populations. It is suggested that this procedure may be useful in the preparation of asymmetric liposomes by facilitating separation of populations devoid of PIP2 in the outer leaflet from unreacted liposomes after enzymatic treatment of symmetric PIP2-containing liposomes.

Topical ciclosporin for psoriasis: in vitro skin penetration and clinical study.

In contrast to oral administration, topical ciclosporin is not effective in the treatment of psoriasis. This may be due to the drug's inability to penetrate the stratum corneum or to a need for systemic metabolism of ciclosporin to active metabolites. We conducted a preliminary evaluation of the ability of ciclosporin, in a variety of vehicles, to penetrate excised human skin in a two-compartment diffusion cell, a standard in vitro technique for evaluating percutaneous drug delivery. A high-pressure liquid chromatography assay for ciclosporin did not detect the passage of ciclosporin through skin. This finding was consistent through multiple trials, some of which evaluated Azone or liposomal formulations, which are reported to enhance the penetrability of certain compounds. These results indicate that ciclosporin is unable to penetrate human skin in the vehicles tested. This may explain the lack of clinical efficacy we demonstrated in 5 patients with psoriasis treated with topical ciclosporin for 14-16 days.

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

The rate of release from multilamellar liposomes of the fluorescent probe carboxyfluorescein was determined as a measure of membrane permeability. Liposomes of phosphatidylcholine and different anionic phospholipids were incubated with low (1 microM) 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 microM 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 microM 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.