Overcoming Semipermeable Barriers, Such as the Skin, with Ultradeformable Mixed Lipid Vesicles, Transfersomes, Liposomes, or Mixed Lipid Micelles Gregor Cevc,* ,† Andreas G. Scha ¨ tzlein, † Holger Richardsen, and Ulrich Vierl IDEA AG, Frankfurter Ring 193a, D-80807 Mu ¨ nchen, Deutschland, EU Received September 20, 2002. In Final Form: August 29, 2003 We studied lipid aggregate penetration through nanoporous, semipermeable barriers by direct transport measurements in vitro and with the confocal laser scanning microscopy of the skin in vivo. We found that it is necessary to use mixed lipid bilayers with a low resistance to permeabilization and high flexibility to overcome narrow, normally confining pores. Partial molecular demixing in the stressed vesicle bilayer serves both purposes. An aggregate comprising a suitable blend of amphipats (Transfersome, Tfs) is, therefore, extremely deformable and easily crosses even very narrow pores (rTfs g 10rpore, and possibly more). Each such vesicle then behaves as a responsive, self-optimizing, nanorobotic transport device. The mixed micelles with identical components or the simple vesicles (liposomes) with a similar size as that of unusually deformable vesicles do not share this quality. Liposomes only traverse barriers when rlipos e 1.5rpore; they clog narrower pores, unless they get fragmented in/before the orifice. Mammalian skin is perforated by a very large number (g10 7 cm -2 ) of very narrow (rpore ∼ 0.3 nm) intercellular hydrophilic pores. These can be widened into the barrier-spanning, hydrophilic transcutaneous pathways (rpathway ∼ 20-30 nm) by ultradeformable vesicles. Mixed micelles or liposomes do not activate such pores because they are respectively too small or too undeformable (κlipos > 10κTfs) and large (2rlipos/nm g 45 . 20) for the purpose. The outer two-thirds of the skin barrier also contain fewer but wider openings (rpore g 3 μm), which encircle groups of cells in the stratum corneum. The resulting sparse, low-resistance intercluster pathway can accommodate various sufficiently small aggregates (ra e 2 μm), including liposomes and micelles. All the tested lipidic particles can, therefore, reach locally ∼60% of the skin barrier depth, on the average. Ultradeformable vesicles move through the skin most uniformly and to the greatest relative depth, however. Locally or near the skin surface the distribution of different lipid aggregates that penetrate a barrier can be similar. 1. Introduction The skin (cutis) is one of the best biological transport barriers. This is mainly due to the outermost layer of the skin, the stratum corneum (see left panel in Figure 1). The latter is 10-30-μm thick 1 and made of stacks of dead or dying keratinized cells, so-called corneocytes. Corneo- cytes form laterally intercalated stacks that are organized in columns (middle panel in Figure 1). Each column is oriented perpendicular to the skin surface and contains a few dozen corneocytes “glued” together with specialized, very hydrophobic lipids. 2 Intercellular lipids in the skin are mainly located in crystalline lipid multilamellae 3 (top- right panel in Figure 1) and are covalently bound to the corneocyte envelope membranes. 4 This increases the skin tightness to small molecules, such as water, and me- chanically strengthens the barrier on a short length scale. To keep the skin flexible on a longer scale, and to allow a good fit between the planar lipids and the imperfectly flat cell envelope membranes, lipid multilamellae are merged through regions of less well-organized lipids, 2 sometimes in all directions. Three to seven adjacent corneocyte columns in the skin typically form a cluster of cells. 8 Adjacent cell groups near the stratum corneum surface are separated by 4-6 μm wide valleys, or clefts. 5 These groves are open and “channel-like” near the skin surface and narrower toward the stratum corneum center. 6 Each cleft, at the bottom, is filled with relatively amorphous lipids, which probably do not match the quality of proper intercellular seals. Lipid packing is generally the densest in the central stratum corneum region. 6 Here, very few, if any, intercorneocyte contacts can reach a width of more than approximately 20 nm. 7 The skin barrier structure described in previous para- graphs suggests that different skin surrogates should be used for in vitro tests. To simulate in the simplest possible fashion the skin permeability barrier, a silastic membrane is often exploited; more trustworthy is the employment of excised skin or of its outermost part, the epidermis, for example, in a “Franz cell”. 8 Nanoporous systems are needed to model the skin penetration barrier and to mimic, at least in the first approximation, the size and shape of pores shown in the right bottom panel of Figure 1. Lipid vesicles, liposomes, were first used as potential (trans)dermal drug carriers in the early 1980s. 9-10 It * Author to whom correspondence should be addressed. † Present address: Cancer Research Unit, Department of Medical Oncology, Garscube Estate, Switchback Rd., Glasgow G61 1BD, U.K. (1) Christophers, E. In The Skin of Vertebrates; Spearman, R. I. C.; Riley, P. A., Eds.; Academic Press: London, 1980; pp 137-139. (2) Wertz, P. W. In Phospholipids: Characterization, Metabolism and Novel Biological Applications; Cevc, G., Paltauf, F., Eds.; AOCS Press: Champaign, 1995; pp 139-158. (3) Bouwstra, J. A.; Goris, G. S.; van der Spek, J. A.; Bras, W. J. Invest. Dermatol. 1991, 97, 1005-1012. (4) Swartzendruber, D. C.; Wertz, P. W.; Madison, K. C.; Downing, D. T. J. Invest. Dermatol. 1987, 88, 709-713. (5) Grove, G. L.; Grove, M. J. In Noninvasive topography assessment in cutaneous investigation in health and disease. Noninvasive methods and instrumentation; Leveque, J.-L., Ed.; Marcel Dekker, Inc.: New York, 1989; pp 1-32. (6) Scha ¨ tzlein, A.; Cevc, G. Br. J. Dermatol. 1998, 138, 583-592. (7) Aguiella, V.; Kontturi, K.; Murtoma ¨ ki, L.; Ramı ´rez, P. J. Controlled Release 1994, 32, 249-257. (8) Hadgraft, J., Guy, R. H., Eds. Transdermal Drug Delivery. Developmental Issues and Research Initiatives; Marcel Dekker: New York, 1989. 10753 Langmuir 2003, 19, 10753-10763 10.1021/la026585n CCC: $25.00 © 2003 American Chemical Society Published on Web 11/21/2003