J, Cosmet. Sci., 52, 265-280 (September/October 2001) ElucidatinD penetration pathways into the hair fiber usinD novel microscopic techniques C. L. GUMMER, Procter & Gamble Technical Centres Ltd., Rusham Park, Whitehall Lane, Egham, S/•rrey, TW20 9NW, United Kingdom. Accepted for publication May 31, 2001. Synopsis Much controversy exists regarding the route of penetration of molecules into hair fibers. In brief, there are two schools of thought. The first argument is that molecules enter the hair fiber via the cell membrane complex (cmc) of the cuticle and then diffuse throughout the cortex via both the intercellular cement and the bulk of individual cortical cells. The second approach concludes that entry to the fiber is via the endocuticle and other non-keratinous parts of the fiber. In tlne latter case the cmc is definitely not considered to have a role in the penetration of molecules into the fiber. The tools available for studying penetration into tlne fiber, e.g., light and electron microscopy, mean that it is usually only possible to extract static information from a dynamic process. Similarly, great care is needed in the interpretation of images produced by the various techniques. Where a molecule is seen to end up does not always indicate how it got there! In these studies I have used novel derivations of conventional electron microscopic techniques, combined with early photographic chemistry, to elucidate further the pathways of penetration into the hair fiber. From these studies one can conclude that both arguments describing penetration into the fiber are complementary, valid, and highly relevant. The techniques allow one to visualize material within the cell membrane complex of the cuticle. In addition, these studies show tinat the high-sulphur proteins of the cuticle, usually considered as highly cross-linked and inaccessible, are easily penetrated. Therefore, all of the structures within a hair fiber should be considered as penetration routes into the hair fiber for the delivery of industrial and cosmetic materials, even though they may not form continuous pathways throughout the hair. The hair should be viewed as a structure composed of a number of compartments of differing capacity, chemistry, and accessibility, ratlner than as continuous pathways from the surface to the center of the fiber. INTRODUCTION Understanding the penetration of molecules into animal fibers is particularly important for the textile dyeing and cosmetic industries. Fiber penetration is an intrinsic compo- nent of dye uptake, dye fastness, cosmetic ingredient penetration, and efficacy. If one knows the route a molecule takes into a fiber and the physicochemical nature of that pathway, then one can better control the penetration of molecules into, and out of, the fiber. Similarly, one can determine the capacity and reactivity of structures in the fiber. Many of the published works consider penetration as the movement of a molecule from a formulation or dye bath into the fiber cortex. This, in turn, implies that the cuticle is 265

266 JOURNAL OF COSMETIC SCIENCE a boundary to be crossed and ignores the important contribution of this layer and its component structures to the desired end result, e.g., a change in hair color. There are currently two hypotheses for the penetration of materials into the hair fiber. Each of these appears to consider the passage of molecules across the cuticle but does not answer important questions regarding fiber structure or the efficacy of formulations. The first hypothesis considers the endocuticle of each cuticle cell to be the main pathway to the cortex (1-3). However, each cuticle cell is bounded by a cell membrane complex both between cells and between the cuticle and cortex. This first approach offers no solid explanation as to how molecules exit the endocuticle and enter the cortex. This would require molecules to pass across the multilamellar cmc, and there is no obvious structural pathway. Mention is made of diffusion via the non-keratinous regions of the fiber, but the cmc is viewed as being physically too small to play a major role in penetration. The second approach considers the cell membrane complex between cuticle cells as the primary route to the cortex (4,5). This is argued on the fact that the cmc of the cuticle, particularly the cmc cement, and cortex are a continuous pathway throughout the fiber. However, this approach does not adequately explain the rapid loss of molecules such as hair dyes from the fiber during routine washing, i.e., how they get out of the cortex and back along the cmc. In these studies I have used novel derivations of established electron microscopy tech- niques. These have been combined with early photographic chemistry to place electron- dense material within hair structures. This can only occur if the molecules have free access into those structures. When studying fiber penetration it is important to under- stand fully the morphology of the fiber. Only then can one begin to interpret the data and understand that the postulated mechanisms are, in fact, complementary. METHODS 1. Using routine light microscopy, single fibers containing a continuous medulla were placed dry under a cover slip. The ends of the fibers protruded from the edges of the cover slip. No mounting media was used. Water was delivered under the cover slip, with continuous observation of the fiber at room temperature. 2. Hair fibres were treated before processing for transmission electron microscopy with one or more of the following reagents: uranyl acetate, lead citrate, aqueous silver nitrate, or ammoniacal silver nitrate (6), all at room temperature. 3. Hairs were immersed in 10% aqueous silver nitrate in the dark for either two minutes or ten minutes at room temperature, rinsed, dried, and then exposed to light. 4. Hair fibers were immersed in aqueous silver nitrate for ten minutes at room tem- perature. The solution was then made alkaline with sodium hydroxide to precipitate silver hydroxide within the fiber also at room temperature. Fibers were then pro- cessed for TEM. 5. Hair fibers were immersed in aqueous sodium chloride (2-60 minutes), rinsed in DI water, and dried. The fibers were then immersed in aqueous silver nitrate (2-60 minutes) to deposit silver chloride within the fiber, rinsed, and dried. The fibers were then exposed to bright light to produce metallic silver inside the fiber and processed for TEM. All steps were performed at room temperature. 6. Processing for TEM: No fixation was used. Fibers were embedded in Spur's low- viscosity resin. For most experiments no post staining of grids was used.