Joke A. Bouwstra

Liposomes and niosomes as topical drug carriers: dermal and transdermal drug delivery

Abstract A critical analysis of (trans) dermal delivery of substances encapsulated within liposomes and niosomes is presented. Topical liposomes or niosomes may serve as solubilization matrix, as a local depot for sustained release of dermally active compounds, as penetration enhancers, or as rate-limiting membrane barrier for the modulation of systemic absorption of drugs. The mechanism(s) of vesicle-skin interaction and drug delivery are being extensively investigated using radioactive- or fluorescence-labeled marker molecules and drugs, and various electron and (laser) light microscopic visualization techniques, and different models describing the interaction with and fate of vesicles in the skin have been proposed. With the current experimental data base on hand, most investigators agree that direct contact between vesicles and skin is essential for efficient delivery, although phospholipids per se apparently do not penetrate into deeper skin layers. Investigators have mostly focused on dermal corticosteroid liposome products. However, localized effects of liposome-associated proteins such as superoxide dismutase, tissue growth factors and interferons appear also to be enhanced. The delivery of liposome-encapsulated proteins and enzymes into deeper skin layers has been reported, although the mechanism of delivery remains to be elucidated. An objective assessment of the performance of topical liposome formulations vs. conventional dosage forms is frequently obscured by investigators comparing equal concentrations, rather than equivalent thermodynamic activities of their respective formulations. We conclude that liposomes and niosomes may become a useful dosage form for a variety of dermally active compounds, specifically due to their ability to modulate drug transfer and serve as nontoxic penetration enhancers.

Chemical enhancement of percutaneous absorption in relation to stratum corneum structural alterations

The outermost layer of the skin, stratum corneum (SC), provides an outstanding barrier against the external environment and is also responsible for skin impermeability toward most solutes. The barrier function is related to the unique composition of the SC lipids and their complex structural arrangement. The lipoidal matrix of the SC, therefore, is a target of penetration enhancer action. The literature on the skin barrier structure and function and on the mechanisms of action of some well established permeation promoters, with a focus on their impact on SC structural alterations, is reviewed. Data obtained from infrared, thermal, and fluorescence spectroscopic examinations of the SC and its components imply enhancer improved permeation of solutes through the SC is associated with alterations involving the hydrocarbon chains of the SC lipid components. Data obtained from electron microscopy and X-ray diffraction reveals that the disordering of the lamellar packing is also an important mechanism for increased permeation of drugs induced by penetration enhancers.

Vesicles as a tool for transdermal and dermal delivery.

Transdermal and dermal drug delivery is problematic because the skin, as a natural barrier, has a very low permeation rate. Therefore several methods have been assessed to increase this rate locally and temporarily. One approach is the use of vesicle formulations. In this paper the effectiveness of conventional and deformable vesicles as drug delivery systems as well as their possible mode of action as permeation enhancers or transdermal drug carriers will be discussed.:

Structure of human stratum corneum as a function of temperature and hydration: A wide-angle X-ray diffraction study

Wide-angle X-ray diffraction measurements were performed as a function of temperature and hydration on human stratum corneum. Reflections located close to the beam stop (d > 1 nm) could be explained on the basis of two lamellar structures. These findings confirmed results obtained with small-angle X-ray diffraction (Bouwstra et al., J. Invest. Dermatol., 97 (1991b) 1005–1012), in which the several complicated diffraction peaks could be ascribed to two lamellar structures with repeat distances of 6.4 and 13.4 nm. In the wide-angle region of the diffraction pattern, we found two diffraction lines, that are characteristic of orthorhombic and hexagonal lateral packing of the lipids. Moreover, the strongest reflections of polycrystalline cholesterol were detected. Upon heating stratum corneum, a phase transition from an orthorhombic to a hexagonal phase in the lateral packing of the lipids at 40°C was observed. Between 60 and 75°C disordering of the lamellar structure occurred, while the hexagonal lateral packing was still present. Between 75 and 95°C, the hexagonal lateral packing disappeared. No changes were found in the reflection lines characteristic of protein. After recrystallisation of the lipids from 120 or 95°C, several higher diffraction orders of the lamellar structure with a repeat distance of 13.4 nm were found. Variation in the hydration level between 6 and 40% w/w did not lead to a shift in the position of the reflections characteristic of lateral packing. In addition, in the recent paper cited above, swelling of the bilayers was not observed, indicating that no water is absorbed between the lamellar regions of human stratum corneum.

Skin structure and mode of action of vesicles

The natural function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. Since the lipids regions in the stratum corneum form the only continuous structure, substances applied onto the skin always have to pass these regions. Therefore, in the first part of this paper, the barrier function has been explained, focusing on the lipid composition and organisation. The major obstacle for topical drug delivery is the low diffusion rate of drugs across the stratum corneum. Several methods have been assessed to increase the permeation rate of drugs temporarily. One of the approaches is the application of drugs in formulations containing vesicles. In order to unravel the mechanisms involved in increasing the drug transport across the skin, information on the effect of vesicles on drug permeation rate, the permeation pathway and perturbations of the skin ultrastructure is of importance. In the second part of this paper, the possible interactions between vesicles and skin are described, focusing on differences between the effects of gel-state, liquid-state, and elastic vesicles.

Modes of action of terpene penetration enhancers in human skin; Differential scanning calorimetry, small-angle X-ray diffraction and enhancer uptake studies

The mechanisms through which the terpenes, d-limonene, 1–8-cineole and nerolidol, increase the permeability of human stratum corneum (s.c.) and the mechanisms underlying propylene glycol (PG)/terpene synergy were investigated using differential scanning calorimetry (DSC), small-angle X-ray diffraction (SAXD) and enhancer uptake studies. DSC experiments identified two major lipid transitions at 72° and 83°C. d-Limonene reduced the temperatures of both transitions by approx. 20°C without affecting their enthalpies (ΔH). 1–8-Cineole also reduced the temperatures of both transitions by approx. 20°C but also reduced ΔH for the first major lipid transition; ΔH for the second was unaffected. d-Limonene increased the combined entropy change (ΔS) associated with both lipid transitions by 11% whereas 1–8-cineole decreased ΔS by 32%. The decrease in ΔS produced by 1–8-cineole provides evidence that this enhancer is lipid disruptive at normal skin temperature. Nerolidol reduced the transition temperatures of both major lipid transitions by approx. 4°C and also decreased their cooperativity. Reduced bilayer cooperativity indicates that this enhancer also disrupts the intercellular lipids. Lack of a clear baseline prevented accurate measurement of ΔH and ΔS values following nerolidol treatment. SAXD experiments showed that d-limonene and 1–8-cineole act to reduce the intensity of lipid based reflections. Decreases in reflection intensities may be linked to a disruption of lipid packing within the bilayers and/or to a disturbance in the stacking of the bilayers. Treatment with nerolidol did not markedly reduce the intensities of the bilayer based reflections. Uptake studies revealed that large quantities of terpenes can be accommodated by the s.c. (mean uptake of d-limonene, 1–8-cineole and nerolidol was 8.90%, 26.2% and 39.6% w/w dry s.c.). The possibility that terpene enhancers pool in the s.c. is discussed. DSC and SAXD investigations provided fragmented evidence that PG/terpene synergy may produce enhanced lipid bilayer disruption. Enhancer uptake studies showed that PG does not significantly increase terpene delivery to the s.c. above that provided by application of neat terpenes.

Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients

A hallmark of atopic eczema (AE) is skin barrier dysfunction. Lipids in the stratum corneum (SC), primarily ceramides, fatty acids, and cholesterol, are crucial for the barrier function, but their role in relation to AE is indistinct. Filaggrin is an epithelial barrier protein with a central role in the pathogenesis of AE. Nevertheless, the precise causes of AE-associated barrier dysfunction are largely unknown. In this study, a comprehensive analysis of ceramide composition and lipid organization in nonlesional SC of AE patients and control subjects was performed by means of mass spectrometry, infrared spectroscopy, and X-ray diffraction. In addition, the skin barrier and clinical state of the disease were examined. The level of ceramides with an extreme short chain length is drastically increased in SC of AE patients, which leads to an aberrant lipid organization and a decreased skin barrier function. Changes in SC lipid properties correlate with disease severity but are independent of filaggrin mutations. We demonstrate for the first time that changes in ceramide chain length and lipid organization are directly correlated with the skin barrier defects in nonlesional skin of AE patients. We envisage that these insights will provide a new therapeutic entry in therapy and prevention of AE.

In vivo assessment of safety of microneedle arrays in human skin.

Microneedle arrays are promising devices for the delivery of drugs and vaccines into or the skin. However, little is known about the safety of the microneedles. In this study we obtained insight in the ability of microneedles to disrupt the skin barrier, which was evaluated by transepidermal water loss (TEWL). We also determined the safety in terms of skin irritation (skin redness and blood flow) and pain sensation. We applied microneedle arrays varying in length and shape on the ventral forearms of 18 human volunteers. An effect of needle length was observed, as TEWL and redness values after treatment with solid microneedle arrays of 400 microm were significantly increased compared to 200 microm. The blood flow showed a similar trend. Needle design also had an effect. Assembled microneedle arrays induced higher TEWL values than the solid microneedle arrays, while resulting in less skin irritation. However, for all microneedles the irritation was minimal and lasted less than 2h. In conclusion, the microneedle arrays used in this study are able to overcome the barrier function of the skin in human volunteers, are painless and cause only minimal irritation. This opens the opportunity for dermal and transdermal delivery of drugs and vaccines.

Elasticity of vesicles assessed by electron spin resonance, electron microscopy and extrusion measurements

The composition of vesicles determines the physical state and elasticity of their bilayers. Fatty acid spin labels were incorporated into vesicles, composed of the single chain non-ionic surfactant octaoxyethylenelaurate-ester (PEG-8-L), the sucrose laurate-ester L-595 and cholesterol sulfate (CS) to monitor local dynamic properties of lipid molecules in vesicle bilayers and to study the elasticity of vesicle bilayers. Studies with the spin label probes 5-, 12- and 16-doxyl stearic acid (DSA) indicated that both the order parameter and the rotational correlation times increased when the doxyl group was positioned closer to the headgroup region. These findings indicate that the fluidity of membranes decreased near the headgroup region. Comparing 16-DSA incorporated in vesicle formulations with either 30 or 70 mol% showed no difference in alkyl chain mobility as was reflected by the order parameter. The rotational correlation times, however, showed a slowdown from 0.38 to 0.71 and 1.13 ns when the PEG-8-L molar content was decreased from 100 to 70 and 30 mol% for PEG-8-L:L-595:CS vesicles, respectively. Extrusion measurements indicated an increase in elasticity of vesicle bilayers as the molar content of PEG-8-L was increased from 10 to 90 mol%. Incorporation of cholesterol sulfate stabilizes vesicles and thereby, decreases the elasticity. The increased elasticity correlated excellent with a reduction in the rotational correlation times observed. In conclusion, these results demonstrate that when the molar content of the single chain non-ionic surfactant PEG-8-L in vesicles is increased the elasticity is enhanced and the rotational correlation time is reduced. The enhanced elasticity might contribute to an optimal design of vesicles as drug carriers for transdermal application.

Administration routes affect the quality of immune responses: A cross-sectional evaluation of particulate antigen-delivery systems

Particulate delivery systems such as liposomes and polymeric nano- and microparticles are attracting great interest for developing new vaccines. Materials and formulation properties essential for this purpose have been extensively studied, but relatively little is known about the influence of the administration route of such delivery systems on the type and strength of immune response elicited. Thus, the present study aimed at elucidating the influence on the immune response when of immunising mice by different routes, such as the subcutaneous, intradermal, intramuscular, and intralymphatic routes with ovalbumin-loaded liposomes, N-trimethyl chitosan (TMC) nanoparticles, and poly(lactide-co-glycolide) (PLGA) microparticles, all with and without specifically selected immune-response modifiers. The results showed that the route of administration caused only minor differences in inducing an antibody response of the IgG1 subclass, and any such differences were abolished upon booster immunisation with the various adjuvanted and non-adjuvanted delivery systems. In contrast, the administration route strongly affected both the kinetics and magnitude of the IgG2a response. A single intralymphatic administration of all evaluated delivery systems induced a robust IgG2a response, whereas subcutaneous administration failed to elicit a substantial IgG2a response even after boosting, except with the adjuvanted nanoparticles. The intradermal and intramuscular routes generated intermediate IgG2a titers. The benefit of the intralymphatic administration route for eliciting a Th1-type response was confirmed in terms of IFN-gamma production of isolated and re-stimulated splenocytes from animals previously immunised with adjuvanted and non-adjuvanted liposomes as well as with adjuvanted microparticles. Altogether the results show that the IgG2a associated with Th1-type immune responses are sensitive to the route of administration, whereas IgG1 response associated with Th2-type immune responses were relatively insensitive to the administration route of the particulate delivery systems. The route of administration should therefore be considered when planning and interpreting pre-clinical research or development on vaccine delivery systems.